Inhibitors of histone deacetylase

ABSTRACT

The invention provides compoods and methods for treating cell proliferative-diseases. The invention provides new inhibitors of histone deacetylase enzymatic activity, compositions of the compounds comprising the inhibitors and a pharmaceutically acceptable carrier, excipient, or diluent, and methods of using the compounds to inhibit cellular proliferation in vitro and therapeutically.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the inhibition of histone deacetylase. Moreparticularly, the invention relates to compounds and methods forinhibiting histone deacetylase enzymatic activity.

2. Summary of the Related Art

In eukaryotic cells, nuclear DNA associates with histones to form acompact complex called chromatin. The histones constitute a family ofbasic proteins which are generally highly conserved across eukaryoticspecies. The core histones, termed H2A, H2B, H3, and H4, associate toform a protein core. DNA winds around this protein core, with the basicamino acids of the histones interacting with the negatively chargedphosphate groups of the DNA. Approximately 146 base pairs of DNA wraparound a histone core to make up a nucleosome particle, the repeatingstructural motif of chromatin.

Csordas, Biochem. J., 286: 23-38 (1990) teaches that histones aresubject to posttranslational acetylation of the α,ε-amino groups ofN-terminal lysine residues, a reaction that is catalyzed by histoneacetyl transferase (HAT1). Acetylation neutralizes the positive chargeof the lysine side chain, and is thought to impact chromatin structure.Indeed, Taunton et al., Science, 272: 408411 (1996), teaches that accessof transcription factors to chromatin templates is enhanced by histonehyperacetylation. Taunton et al. further teaches that an enrichment inunderacetylated histone H4 has been found in transcriptionally silentregions of the genome.

Histone acetylation is a reversible modification, with deacetylationbeing catalyzed by a family of enzymes termed histone deacetylases(HDACs). Grozinger et al., Proc. Natl. Acad. Sci. USA, 96: 4868-4873(1999), teaches that HDACs is divided into two classes, the firstrepresented by yeast Rpd3-like proteins, and the second represented byyeast Hda1-like proteins. Grozinger et al. also teaches that the humanHDAC1, HDAC2, and HDAC3 proteins are members of the first class ofHDACs, and discloses new proteins, named HDAC4, HDAC5, and HDAC6, whichare members of the second class of HDACs. Kao et al., Genes & Dev., 14:55-66 (2000), discloses HDAC7, a new member of the second class ofHDACs. Van den Wyngaert, FEBS, 478: 77-83 (2000) discloses HDAC8, a newmember of the first class of HDACs.

Richon et al., Proc. Natl. Acad. Sci. USA, 95: 3003-3007 (1998),discloses that HDAC activity is inhibited by trichostatin A (TSA), anatural product isolated from Streptomyces hygroscopius, and by asynthetic compound, suberoylanilide hydroxamic acid (SAHA). Yoshida andBeppu, Exper. Cell Res., 177: 122-131 (1988), teaches that TSA causesarrest of rat fibroblasts at the G₁ and G₂ phases of the cell cycle,implicating HDAC in cell cycle regulation. Indeed, Finnin et al.,Nature, 401: 188-193 (1999), teaches that TSA and SAHA inhibit cellgrowth, induce terminal differentiation, and prevent the formation oftumors in mice. Suzuki et al., U.S. Pat. No. 6,174,905, EP 0847992, JP258863/96, and Japanese Application No. 10138957, disclose benzamidederivatives that induce cell differentiation and inhibit HDAC. Delormeet al., WO 01/38322 and PCT IB01/00683, disclose additional compoundsthat serve as HDAC inhibitors.

These findings suggest that inhibition of HDAC activity represents anovel approach for intervening in cell cycle regulation and that HDACinhibitors have great therapeutic potential in the treatment of cellproliferative diseases or conditions. To date, few inhibitors of histonedeacetylase are known in the art. There is thus a need to identifyadditional HDAC inhibitors and to identify the structural featuresrequired for potent HDAC inhibitory activity.

BRIEF SUMMARY OF THE INVENTION

The invention provides compounds and methods for treating cellproliferative diseases. The invention provides new inhibitors of histonedeacetylase enzymatic activity.

In a first aspect, the invention provides compounds that are useful asinhibitors of histone deacetylase.

In a second aspect, the invention provides a pharmaceutical compositioncomprising an inhibitor of histone deacetylase according to theinvention and a pharmaceutically acceptable carrier, excipient, ordiluent.

In a third aspect, the invention provides a method of inhibiting histonedeacetylase in a cell, comprising contacting a cell in which inhibitionof histone deacetylase is desired with an inhibitor of histonedeacetylase of the invention.

The foregoing merely summarizes certain aspects of the invention and isnot intended to be limiting in nature. These aspects and other aspectsand embodiments are described more fully below.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 displays antineoplastic effects of a histone deacetylaseinhibitor according to the invention on human tumor xenografts in vivo,as described in Example 51, infra.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides compounds and methods for inhibiting histonedeacetylase enzymatic activity. The invention also provides compositionsand methods for treating cell proliferative diseases and conditions. Thepatent and scientific literature referred to herein establishesknowledge that is available to those with skill in the art. The issuedpatents, applications, and references that are cited herein are herebyincorporated by reference to the same extent as if each was specificallyand individually indicated to be incorporated by reference. In the caseof inconsistencies, the present disclosure will prevail.

In one embodiment of the first aspect, the invention comprises compoundsof the following formula:

and pharmaceutically acceptable salts thereof, wherein

Ar is aryl or heteroaryl, each of which is optionally substituted withfrom 1 to 3 substituents.

Preferably, Ar is aryl or pyridinyl in the compound of paragraph [0014].

Preferred substituents of Ar include halo, C₁-C₆-hydrocarbyl optionallysubstituted with halo, C₁-C₆-hydrocarbyloxy optionally substituted withhalo. Particularly preferred substituents include fluoro, chloro,methoxy, cyclopropyloxy, and cyclopentyloxy.

In a preferred embodiment of the compound according to paragraph [0014],Ar is selected from the following:

Preferred compounds of paragraph [0014] include those of Table 6 below.

In another embodiment of the first aspect, the invention comprisescompounds of the following formula:

and pharmaceutically acceptable salts thereof, wherein

X is —N(R¹)—, —O—, or —S—; or X is a nitrogen-containing heterocyclyl inwhich a nitrogen is covalently bound to the adjacent carbonyl instructure V and is optionally substituted with from 1 to 3 substituents;and

R and R¹ independently are —H, or optionally substituted a)C₁-C₆-hydrocarbyl or b) R²-L-, wherein R² is aryl or heteroaryl, L isC₀-C₆-hydrocarbyl-L¹-C₀-C₆-hydrocarbyl, and L¹ is a covalent bond, —O—,—S—, or —NH—.

Preferably in the compound according to paragraph [0019], X is —NH—,—O—, morphilin-4-yl, piperidin-1-yl, piperizin-1-yl, or pyrrolidin-1-yl.

In another preferred embodiment of the compound according to paragraph[0019], X is —N(R¹)— wherein R¹ is optionally substituted methyl orethyl. Preferably R¹ is cyanoethyl or pyridinylmethyl.

Preferably in the compound according to paragraph [0019], R is R²-L-wherein R² is phenyl, pyridinyl, indyl, or indolyl and L is a covalentbond, methyl, ethyl, or oxyethyl.

Preferred subsbtuents of R include methoxy and hydroxy.

In a preferred embodiment of the compound according to paragraph [0019],the combination of R—X— is selected from the following:

Preferred compounds according to paragraph [0019] include those listedin Table 7.

In another embodiment of the first aspect, the invention comprisescompounds of the following formula:

and pharmaceutically acceptable salts thereof, wherein

Y is —N(R⁴)—, —O—, —S—, —N(R⁴)SO₂—, —SO₂—N(R⁴)—, —SO₂—, —N(R⁴)—C(O)—,—C(O)—N(R⁴)—, —NHC(O)NH—, —N(R⁴)C(O)O—, —OC(O)N(R⁴)—, or a covalentbond, and

R¹, R², and R³ independently are —H or R^(a)—C₀-C₆-hydrocarbyl whereinR^(a) is —H or R^(a) is aryl or heteroaryl, each of which is optionallysubstituted with from 1 to 3 substituents.

R⁴ is —H, —C(O)—R^(b), —C(O)O—R^(b), —C(O)NH—R^(b), orR^(c)—C₀-C₆-hydrocarbyl wherein

R^(b) is —H or —C₁-C₆-hydrocarbyl, and

R^(c) is —H, or aryl or heteroaryl each of which is optionallysubstituted with from 1 to 3 substituents.

In the compound of paragraph [0026], R² and R³ are preferably both —H.

In the compound of paragraph [0026], Y is preferably —NH—, —SO₂NH—, or—N(R⁴)— wherein R⁴ is —C(O)O—C₁-C₆-hydrocarbyl.

In the compound of paragraph [0026], R¹ is preferably aryl,benzothiazolyl, pyrimidinyl, triazolyl, benzodioxolenyl, or pyridinyl.

Preferred substituents of R¹ include C₁-C₆-hydrocarbyl,C₁-C₆-hydrocarbyloxy (e.g., methoxy and cyclopropyloxy) halo,methylthio, and acetyl.

In a preferred embodiment of the compound according to paragraph [0026],R¹—Y— is selected from the following:

Preferred compounds according to paragraph [0026] include those listedin Table 8.

In another embodiment of the first aspect, the invention comprisescompounds of formula:

and pharmaceutically acceptable salts thereof, wherein Ar¹ is aryl orheteroaryl optionally substituted with from 1-3 substituentsindependently selected from —NO₂, CH₃O—, and morpholinyl (e.g.,morpholin-4-yl).

In a preferred embodiment of the compound according to paragraph [0033],Ar¹ is aryl (e.g., phenyl).

In preferred embodiments of the compound according to paragraph [0033],Ar¹ is selected from:

Preferred compounds according to paragraph [0033] included those listedin Table 9.

In the second aspect, the invention comprises a composition comprising acompound according to one of paragraphs [0014]-[0036] and apharmaceutically acceptable carrier, excipient, or diluent.

In a third aspect, the invention provides a method of inhibiting histonedeacetylase in a cell, comprising contacting a cell in which inhibitionof histone deacetylase is desired with an inhibitor of histonedeacetylase according to one of paragraphs [0014]-[0037].

In another aspect, the invention comprises treating a mammal (preferablya human) suffering from a cell proliferative diseases or conditions atherapeutically effective amount of a composition according to paragraph[0037].

For purposes of the present invention, the following definitions will beused (unless expressly stated otherwise):

As used herein, the terms “histone deacetylase” and “HDAC” are intendedto refer to any one of a family of enzymes that remove acetyl groupsfrom the, -amino groups of lysine residues at the N-terminus of ahistone. Unless otherwise indicated by context, the term “histone” ismeant to refer to any histone protein, including H1, H2A, H2B, H3, H4,and H5, from any species. Preferred histone deacetylases include class Iand class II enzymes. Preferably the histone deacetylase is a humanHDAC, including, but not limited to, HDAC-1, HDAC-2, HDAC-3, HDAC-4,HDAC-5, HDAC-6, HDAC-7, and HDAC-8. In some other preferred embodiments,the histone deacetylase is derived from a protozoal or fungal source.

The terms “histone deacetylase inhibitor” and “inhibitor of histonedeacetylase” are used to identify a compound having a structure asdefined herein, which is capable of interacting with a histonedeacetylase and inhibiting its enzymatic activity. “Inhibiting histonedeacetylase enzymatic activity” means reducing the ability of a histonedeacetylase to remove an acetyl group from a histone. In some preferredembodiments, such reduction of histone deacetylase activity is at leastabout 50%, more preferably at least about 75%, and still more preferablyat least about 90%. In other preferred embodiments, histone deacetylaseactivity is reduced by at least 95% and more preferably by at least 99%.

Preferably, such inhibition is specific, i.e., the histone deacetylaseinhibitor reduces the ability of a histone deacetylase to remove anacetyl group from a histone at a concentration that is lower than theconcentration of the inhibitor that is required to produce another,unrelated biological effect. Preferably, the concentration of theinhibitor required for histone deacetylase inhibitory activity is atleast 2-fold lower, more preferably at least 5-fold lower, even morepreferably at least 10-fold lower, and most preferably at least 20-foldlower than the concentration required to produce an unrelated biologicaleffect.

For simplicity, chemical moieties are defined and referred to throughoutprimarily as univalent chemical moieties (e.g., alkyl, aryl, etc.).Nevertheless, such terms are also used to convey correspondingmultivalent moieties under the appropriate structural circumstancesclear to those skilled in the art. For example, while an “alkyl” moietygenerally refers to a monovalent radical (e.g. CH₃—CH₂—), in certaincircumstances a bivalent linking moiety can be “alkyl,” in which casethose skilled in the art will understand the alkyl to be a divalentradical (e.g., —CH₂—CH₂—), which is equivalent to the term “alkylene.”(Similarly, in circumstances in which a divalent moiety is required andis stated as being “aryl,” those skilled in the art will understand thatthe term “aryl” refers to the corresponding divalent moiety, arylene.)All atoms are understood to have their normal number of valences forbond formation (i.e., 4 for carbon, 3 for N. 2 for O, and 2, 4, or 6 forS, depending on the oxidation state of the S). On occasion a moiety maybe defined, for example, as (A)_(a)-B—, wherein a is 0 or 1. In suchinstances, when a is 0 the moiety is B— and when a is 1 the moiety isA-B—. Also, a number of moieties disclosed herein exist in multipletautomeric forms, all of which are intended to be encompassed by anygiven tautomeric structure.

The term “hydrocarbyl” refers to a straight, branched, or cyclic alkyl,alkenyl, or alkynyl, each as defined herein. A “C₀” hydrocarbyl is usedto refer to a covalent bond. Thus, “C₀-C₃-hydrocarbyl” includes acovalent bond, methyl, ethyl, ethenyl, ethynyl, propyl, propenyl,propynyl, and cyclopropyl.

The term “alkyl” as employed herein refers to straight and branchedchain aliphatic groups having from 1 to 12 carbon atoms, preferably 1-8carbon atoms, and more preferably 1-6 carbon atoms, which is optionallysubstituted with one, two or three substituents. Preferred alkyl groupsinclude, without limitation, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl. A “C₀” alkyl (as in“C₀-C₃-alkyl”) is a covalent bond (like “C₀” hydrocarbyl).

The term “alkenyl” as used herein means an unsaturated straight orbranched chain aliphatic group with one or more carbon-carbon doublebonds, having from 2 to 12 carbon atoms, preferably 2-8 carbon atoms,and more preferably 2-6 carbon atoms, which is optionally substitutedwith one, two or three substituents. Preferred alkenyl groups include,without limitation, ethenyl, propenyl, butenyl, pentenyl, and hexenyl.

The term “alkynyl” as used herein means an unsaturated straight orbranched chain aliphatic group with one or more carbon-carbon triplebonds, having from 2 to 12 carbon atoms, preferably 2-8 carbon atoms,and more preferably 2-6 carbon atoms, which is optionally substitutedwith one, two or three substituents. Preferred alkynyl groups include,without limitation, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

An “alkylene,” “alkenylene,” or “alkynylene” group is an alkyl, alkenyl,or alkynyl group, as defined hereinabove, that is positioned between andserves to connect two other chemical groups. Preferred alkylene groupsinclude, without limitation, methylene, ethylene, propylene, andbutylene. Preferred alkenylene groups include, without limitation,ethenylene, propenylene, and butenylene. Preferred alkynylene groupsinclude, without limitation, ethenylene, propenylene, and butynylene.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally is optionally substituted. Preferredcycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroalkyl” refers to an alkyl group, as defined hereinabove,wherein one or more carbon atoms in the chain are replaced by aheteroatom selected from the group consisting of O, S, and N.

An “aryl” group is a C₆-C₁₄ aromatic moiety comprising one to threearomatic rings, which is optionally substituted. Preferably, the arylgroup is a C₆-C₁₀ aryl group. Preferred aryl groups include, withoutlimitation, phenyl, naphthyl, anthracenyl, and fluorenyl. An “aralkyl”or “arylalkyl” group comprises an aryl group covalently linked to analkyl group, either of which may independently be optionally substitutedor unsubstituted. Preferably, the aralkyl group is(C₁-C₆)alk(C₆-C₁₀)aryl, including, without limitation, benzyl,phenethyl, and naphthylmethyl.

A “heterocyclyl” or “heterocyclic” group is a ring structure having fromabout 3 to about 12 atoms, wherein one or more atoms are selected fromthe group consisting of N, O, and S. The heterocyclic group isoptionally substituted on carbon at one or more positions. Theheterocyclic group is also independently optionally substituted onnitrogen with alkyl, aryl, aralkyl, alkylcarbonyl, alkylsulfonyl,arylcarbonyl, arylsulfonyl, alkoxycarbonyl, aralkoxycarbonyl, or onsulfur with oxo or lower alkyl. Preferred heterocyclic groups include,without limitation, epoxy, aziridinyl, tetrahydrofuranyl, pyrrolidinyl,piperidinyl, piperazinyl, thiazolidinyl, oxazolidinyl, oxazolidinonyl,and morpholino. In certain preferred embodiments, the heterocyclic groupis fused to an aryl, heteroaryl, or cycloalkyl group. Examples of suchfused heterocyles include, without limitation, tetrahydroquinoline anddihydrobenzofuran. Specifically excluded from the scope of this term arecompounds having an annular O and/or S atom adjacent to another annularO or S.

As used herein, the term “heteroaryl” refers to groups having 5 to 14ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14 πelectrons shared in a cyclic array; and having, in addition to carbonatoms, from one to three heteroatoms per ring selected from the groupconsisting of N, O, and S. The term “heteroaryl” is also meant toencompass monocyclic and bicyclic groups. For example, a heteroarylgroup may be pyrimidinyl, pyridinyl, benzimidazolyl, thienyl,benzothiazolyl, benzofuranyl and indolinyl. A “heteroarylalkyl” or“heteroarylalkyl” group comprises a heteroaryl group covalently linkedto an alkyl group, either of which is independently optionallysubstituted or unsubstituted. Preferred heteroalkyl groups comprise aC₁-C₆ alkyl group and a heteroaryl group having 5, 6, 9, or 10 ringatoms. Specifically excluded from the scope of this term are compoundshaving adjacent annular O and/or S atoms. Examples of preferredheteroarylalkyl groups include pyridylmethyl, pyridylethyl,pyrrolylmethyl, pyrrolylethyl, imidazolylmethyl, imidazolylethyl,thiazolylmethyl, and thiazolylethyl. Specifically excluded from thescope of this term are compounds having adjacent annular O and/or Satoms.

An “arylene,” “heteroarylene,” or “heterocyclylene” group is an aryl,heteroaryl, or heterocyclyl group, as defined hereinabove, that ispositioned between and serves to connect two other chemical groups.

Preferred heterocyclyls and heteroaryls include, but are not limited to,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl,carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl,cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isochromanyl,isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl,isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, andxanthenyl.

As employed herein, when a moiety (e.g., cycloalkyl, hydrocarbyl, aryl,heteroaryl, heterocyclic, urea, etc.) is described as “optionallysubstituted” it is meant that the group optionally has from one to four,preferably from one to three, more preferably one or two, non-hydrogensubstituents. Suitable substituents include, without limitation, halo,hydroxy, oxo (e.g., an annular —CH— substituted with oxo is —C(O)—)nitro, halohydrocarbyl, hydrocarbyl, aryl, aralkyl, alkoxy, aryloxy,amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, acyl,carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido,arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, andureido groups. Preferred substituents, which are themselves not furthersubstituted (unless expressly stated otherwise) are:

-   -   (a) halo, cyano, oxo, carboxy, formyl, nitro, amino, amidino,        guanidino,    -   (b) C₁-C₅ alkyl or alkenyl or arylalkyl imino, carbamoyl, azido,        carboxamido, mercapto, hydroxy, hydroxyalkyl, alkylaryl,        arylalkyl, C₁-C₈ alkyl, C₁-C₈ alkenyl, C₁-C₈ alkoxy, C₁-C₈        alkoxycarbonyl, aryloxycarbonyl, C₂-C₈ acyl, C₂-C₈ acylamino,        C₁-C₈ alkylthio, arylalkylthio, arylthio, C₁-C₈ alkylsulfinyl,        arylalkylsulfinyl, arylsulfinyl, C₁-C₈ alkylsulfonyl,        arylalkylsulfonyl, arylsulfonyl, C₀-C₆ N-alkyl carbamoyl, C₂-C₁₅        N,N-dialkylcarbamoyl, C₃-C₇ cycloalkyl, aroyl, aryloxy,        arylalkyl ether, aryl, aryl fused to a cycloalkyl or heterocycle        or another aryl ring, C₃-C₇ heterocycle, or any of these rings        fused or spiro-fused to a cycloalkyl, heterocyclyl, or aryl,        wherein each of the foregoing is further optionally substituted        with one more moieties listed in (a), above; and    -   (c) —(CH₂)_(s)—NR³⁰R³¹, wherein s is from 0 (in which case the        nitrogen is directly bonded to the moiety that is substituted)        to 6, and R³⁰ and R³¹ are each independently hydrogen, cyano,        oxo, carboxamido, amidino, C₁-C₈ hydroxyalkyl, C₁-C₃ alkylaryl,        aryl-C₁-C₃ alkyl, C₁-C₈ alkyl, C₁-C₈ alkenyl, C₁-C₈ alkoxy,        C₁-C₈ alkoxycarbonyl, aryloxycarbonyl, aryl-C₁-C₃        alkoxycarbonyl, C₂-C₈ acyl, C₁-C₈ alkylsulfonyl,        arylalkylsulfonyl, arylsulfonyl, aroyl, aryl, cycloalkyl,        heterocyclyl, or heteroaryl, wherein each of the foregoing is        further optionally substituted with one more moieties listed in        (a), above; or

R³⁰ and R³¹ taken together with the N to which they are attached form aheterocyclyl or heteroaryl, each of which is optionally substituted withfrom 1 to 3 substituents from (a), above.

In addition, substituents on cyclic moieties (i.e., cycloalkyl,heterocyclyl, aryl, heteroaryl) include 5-6 membered mono- and 9-14membered bi-cyclic moieties fused to the parent cyclic moiety to form abi- or tricyclic fused ring system. For example, an optionallysubstituted phenyl includes the following:

A “halohydrocarbyl” is a hydrocarbyl moiety in which from one to allhydrogens have been replaced with one or more halo.

The term “halogen” or “halo” as employed herein refers to chlorine,bromine, fluorine, or iodine. As herein employed, the term “acyl” refersto an alkylcarbonyl or arylcarbonyl substituent. The term “acylamino”refers to an amide group attached at the nitrogen atom (i.e., R—CO—NH—).The term “carbamoyl” refers to an amide group attached at the carbonylcarbon atom (i.e., NH₂—CO—). The nitrogen atom of an acylamino orcarbamoyl substituent is additionally substituted. The term“sulfonamido” refers to a sulfonamide substituent attached by either thesulfur or the nitrogen atom. The term “amino” is meant to include NH₂,alkylamino, arylamino, and cyclic amino groups. The term “ureido” asemployed herein refers to a substituted or unsubstituted urea moiety.

The term “radical” as used herein means a chemical moiety comprising oneor more unpaired electrons.

A moiety that is substituted is one in which one or more hydrogens havebeen independently replaced with another chemical substituent. As anon-limiting example, substituted phenyls include 2-fluorophenyl,3,4-dichlorophenyl, 3chloro-4-fluoro-phenyl, 2-fluor-3-propylphenyl. Asanother non-limiting example, substituted n-octyls include 2,4dimethyl-5-ethyl-octyl and 3-cyclopentyl-octyl. Included within thisdefinition are methylenes (—CH₂—) substituted with oxygen to formcarbonyl —CO—).

An “unsubstituted” moiety as defined above (e.g., unsubstitutedcycloalkyl, unsubstituted heteroaryl, etc.) means that moiety as definedabove that does not have any of the optional substituents for which thedefinition of the moiety (above) otherwise provides. Thus, for example,while an “aryl” includes phenyl and phenyl substituted with a halo,“unsubstituted aryl” does not include phenyl substituted with a halo.

Preferred embodiments of the invention also include combinations of thepreferred embodiments expressly described herein.

Synthesis

Compounds of general formula I were prepared according to the syntheticroutes depicted in Schemes 1 and 2. In some embodiments, 4-acetylbenzoicacid was reacted with an aromatic and/or heteroaromatic aldehyde in asolvent such as methanol (MeOH) in the presence of an aqueous solutionof sodium hydroxide (1N) to give after filtration or acidification untilpH=5-6 and filtration, the chalcone II. Compound II was first treatedwith the coupling reagentbenzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP reagent) in a solvent such as N,N-dimethylformamide (DMF) in thepresence of triethylamine (Et₃N). The resulting activated esterintermediate formed in situ was finally reacted with1,2-phenylenediamine to afford the compound I (Scheme 1).

Alternatively, in some other embodiments, 4-acetylbenzoic acid was firsttreated with the coupling reagentbenzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphatein a solvent such as N,N-dimethylformamide in the presence oftriethylamine. The resulting activated ester intermediate formed in situwas then reacted with t-butyl (2-amino-phenyl)-carbamate to afford thecommon acetophenone derivative III. Chalcone IV was prepared by theClaisen-Schmidt condensation of compound III with an appropriatearomatic and/or heteroaromatic aldehyde in a solvent such as methanol inthe presence of an aqueous solution of sodium hydroxide (1N). N-Bocprotective group of aniline IV was finally cleaved by a wet solution oftrifluoroacetic acid (TFA 95% in water) in a solvent such asdichloromethane (CH₂Cl₂) to furnish the compound I (Scheme 2).

Compounds of general formula V were prepared according to the syntheticroutes depicted in Scheme 3 and 4. In certain preferred embodiments,methyl 4-formylbenzoate was converted into the puretrans-α,β-unsaturated ester VI by reaction with the anion of t-butylacetate in a mixture of solvent such as tetrahydrofuran (THF) and hexanefollowed by the treatment with 2-chloro-4,6-dimethoxy-1,3,5-triazine asa new dehydrating agent. Acidic hydrolysis of t-butyl ester VI wasperformed by a wet solution of trifluoroacetic acid (95% in water) in asolvent such as dichloromethane to give the compound VII. The formationof compounds IX was carried out by two complementary methods dependingof the nucleophilicity of RXH. In the method A, the carboxylic acid VIIwas first converted into the stable activated ester VIII by using thecoupling reagent benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP reagent) in a solvent such asN,N-dimethylformamide in the presence of triethylamine. This stableactivated ester VIII was then reacted with a weak nucleophile (e.g.,RXH=anilines or aminoheteroaryls) in a solvent such as dichloromethanein the presence of triethylamine to afford the compound IX. In themethod B, the same activated ester VIII intermediate formed in situ fromthe carboxylic acid VII, was then reacted with a strong nucleophile(e.g., RXH=amines, alcohols, thiols, hydroxylamine and derivatives, orhydrazine and derivatives) in a solvent such as N,N-dimethylformamide inthe presence of triethylamine to afford the compound IX.

Basic hydrolysis of methyl ester IX was performed by a aqueous solutionof lithium hydroxide in a solvent such as tetrahydrofuran to lead to thecompound X. Carboxylic acid X was finally treated with the couplingreagent benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP reagent) in a solvent such asN,N-dimethylformamide in the presence of triethylamine. The resultingactivated ester intermediate formed in situ was then reacted with1,2-phenylenediamine to afford the compound V (Scheme 3).

Alternatively, in some other embodiments, 4-carboxybenzaldehyde wasfirst converted into the acid chloride intermediate by using thionylchloride (SOCl₂) in a solvent such as dichloromethane in the presence ofa catalytic amount of N,N-dimethylformamide. The resulting acid chlorideintermediate was then reacted with t-butyl (2-amino-phenyl)-carbamate toafford the common benzaldehyde derivative XI.

Wittig olifination of the aldehyde XI was performed with methyl(triphenyl-phosphoranylidene)acetate in a solvent such as toluene toprovide the trans-α,β-unsaturated ester XII. Basic hydrolysis of methylester XII was performed by a aqueous solution of lithium hydroxide in asolvent such as tetrahydrofuran to lead to the compound XIII. Carboxylicacid XIII was treated with the coupling reagentbenzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP reagent) in a solvent such as N,N-dimethylformamide in the presenceof triethylamine. The resulting activated ester intermediate formed insitu was then reacted with the nucleophile R¹XIV to afford the compoundXIV. N-Boc protective group of aniline XIV was finally cleaved by a wetsolution of trifluoroacetic acid (95% in water) in a solvent such asdichloromethane to furnish the compound V (Scheme 4).

Compounds of general formula XV were prepared according to the syntheticroutes depicted in Schemes 5-7. Thus, Wittig olifination of aldehyde XIwas performed with the (triphenylphosphoranylidene)-acetaldehyde reagentin a solvent such as toluene to provide the trans-α,β-unsaturatedaldehyde XVI. In certain preferred embodiments, the formation ofcompounds XVII was performed by a reductive amination following thedescribed method depending of the nucleophilicity of R¹XVI. Aldehyde XVIwas first mixed with a weak nucleophile (e.g., R¹XH=anilines oraminoheteroaryls) in a solvent such as tetrahydrofuran in the presenceof a catalytic amount of dibutyltin dichloride. The resulting iminiumintermediate formed in situ was then reacted with the reductive reagentphenylsilane to afford the compound XVII.

N-Boc protective group of aniline XVII was finally cleaved by a wetsolution of trifluoroacetic acid (95% in water) in a solvent such asdichloromethane to furnish the compound XV (Scheme 5).

Alternatively, in some other embodiments, the trans-α,β-unsaturatedaldehyde XVI was reduced into the primary allylic alcohol XVIII by thereductive reagent sodium borohydride in a solvent such as ethanol. Thisalcohol XVIII was then reacted with a nucleophile R¹XH according to aMitsunobu type reaction in a solvent such as tetrahydrofuran in thepresence of triphenylphosphine and diethyl azodicarboxylate (DEAD) tofurnish the compound XVII. N-Boc protective group of aniline XVII wasfinally cleaved by a wet solution of trifluoroacetic acid (95% in water)in a solvent such as dichloromethane to furnish the compound XV (Scheme6).

Moreover, in some other embodiments, Wittig olefination of methyl4-formylbenzoate was performed using either the(triphenylphosphoranylidene)-acetaldehyde reagent in a solvent such astoluene or the (1,3-dioxolan-2-yl)methyltriphenylphosphonium bromidereagent in the presence of TDA-1 {tris[2-(2-methoxyethoxy)ethyl]amine)and potassium carbonate in a biphasic medium such asdichloromethane/water followed by an acidic hydrolysis to provide thetrans-α,β-unsaturated aldehyde XIX. Aldehyde XIX was first mixed with anucleophile (R¹R²NH) in a solvent such as dichloromethane or1,2-dichloroethane. The resulting iminium intermediate formed in situwas then reacted with the reductive reagent sodium triacetoxyborohydride[NaBH(OAc)₃] to afford the compound XX. In the pathway A, the basichydrolysis of the methyl ester and protection of the secondary amine ofcompound XX (R¹=alkyl, R²=H) were performed at the same time in thepresence of an aqueous solution of sodium hydroxide (1N) and theprotective reagent di-tert-butyl dicarbonate [(Boc)₂O] in a solvent such1,4-dioxane to lead to the compound XXI. Carboxylic acid XXI was firsttreated with the coupling reagentbenzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP reagent) in a solvent such as N,N-dimethylformamide in the presenceof triethylamine. The resulting activated ester intermediate formed insitu was then reacted with 1,2-phenylenediamine to afford the compoundXXII. N-Boc protective group of amine XXII was finally cleaved by asolution of wet trifluoroacetic acid (95% in water) in a solvent such asdichloromethane to furnish the compound XV (Scheme 7).

In the pathway B, the methyl ester XX was directly converted into thefinal compound XV after basic hydrolysis and coupling with1,2-phenylenediamine (Scheme 7).

Compounds of general formula XXIV were prepared according to thesynthetic routes depicted in Schemes 8 and 9. In some embodiments,4-formylbenzoic acid was reacted with an aryl and/or heteroaryl methylketone in a solvent such as methanol (MeOH) in the presence of anaqueous solution of sodium hydroxide (1N) to give after filtration thechalcone XXIII. Compoud XXIII was first treated with the couplingreagent benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP reagent) in a solvent such asN,N-dimethylformamide (DMF) in the presence of triethylamine (Et₃N). Theresulting activated ester intermediate formed in situ was finallyreacted with 1,2-phenylenediamine to afford the compound XXIV (Scheme8).

Alternatively, in some other embodiments, chalcone XXV was prepared bythe Claisen-Schmidt condensation of benzaldehyde derivative XI with anappropriate aryl and/or heteroaryl methyl ketone in a solvent such asmethanol in the presence of an aqueous solution of sodium hydroxide(1N). N-Boc protective group of aniline XXV was finally cleaved by a wetsolution of trifluoroacetic acid (TFA 95% in water) in a solvent such asdichloromethane (CH₂Cl₂) to furnish the compound XXIV (Scheme 9).

Compounds of general formula XXVII and XXIX were prepared according tothe synthetic routes depicted in Scheme 10. Thus, selective reduction ofthe double bond of compound XXIII was carried out by using the reductivereagent benzenesulfonyl hydrazide in a solvent such asN,N-dimethylformamide to produce compound XXVI. Carboxylic acid XXVI wasfirst treated with the coupling reagentbenzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP reagent) in a solvent such as N,N-dimethylformamide in the presenceof triethylamine. The resulting activated ester intermediate formed insitu was finally reacted with 1,2-phenylenediamine to afford thecompound XXVII.

Moreover, the complete reduction of the α,β-unsaturated ketone XXIIIinto the saturated compound XXVIII was performed by an hydrogenationcatalyzed by 10% of palladium on charcoal (Degussa type) in a solventsuch as N,N-dimethylacetamide (DMA). Then, the carboxylic acid XXVIIIwas first treated with the coupling reagentbenzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP reagent) in a solvent such as N,N-dimethylformamide in the presenceof triethylamine. The resulting activated ester intermediate formed insitu was finally reacted with 1,2-phenylenediamine to afford thecompound XXIX (Scheme 10).

Compounds of general formula XXX were prepared according to thesynthetic route depicted in Scheme 11. Thus, Sonogashira type reactionbetween methyl 4-bromobenzoate and (trimethylsilyl)acetylene was carriedout by a catalytic amount of palladium catalyst and copper iodide in thepresence of Et₃N in a solvent such THF to afford the protected alkyneXXXI. Basic deprotecton of TMS group of XXXI was performed by potassiumcarbonate in the presence of methanol to give the alkyne XXXII.Hydroboration of the triple bond of XXXII was performed by thecatecholborane reagent in a solvent such THF followed by acidichydrolysis of the boronate intermediate to furnish boronic acid XXXIII.Allylic amine XXXIV was elaborated according to the Petasis typereaction by reacting the vinylboronic acid XXXIII with a pre-formedmixture between an amino compound (R¹R²NH) and an aldehyde (R³CHO) in asolvent such 1,4-dioxane. Finally, the methyl ester XXIV was convertedinto the final compound XXX after basic hydrolysis and coupling with1,2-phenylenediamine.

Inhibition of Histone Deacetylase

In a third aspect, the invention provides a method of inhibiting histonedeacetylase in a cell, comprising contacting a cell in which inhibitionof histone deacetylase is desired with an inhibitor of histonedeacetylase according to the invention.

Measurement of the enzymatic activity of a histone deacetylase can beachieved using known methodologies. For example, Yoshida et al., J.Biol. Chem., 265: 17174-17179 (1990), describes the assessment ofhistone deacetylase enzymatic activity by the detection of acetylatedhistones in trichostatin A treated cells. Taunton et al., Science, 272:408-411 (1996), similarly describes methods to measure histonedeacetylase enzymatic activity using endogenous and recombinant HDAC-1.

In some preferred embodiments, the histone deacetylase inhibitorinteracts with and reduces the activity of all histone deacetylases inthe cell. In some other preferred embodiments according to this aspectof the invention, the histone deacetylase inhibitor interacts with andreduces the activity of fewer than all histone deacetylases in the cell.In certain preferred embodiments, the inhibitor interacts with andreduces the activity of one histone deacetylase (e.g., HDAC-1), but doesnot interact with or reduce the activities of other histone deacetylases(e.g., HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, and HDAC-8). Asdiscussed below, certain particularly preferred histone deacetylaseinhibitors are those that interact with, and reduce the enzymaticactivity of, a histone deacetylase that is involved in tumorigenesis.Certain other preferred histone deacetylase inhibitors interact with andreduce the enzymatic activity of a fungal histone deacetylase.

Preferably, the method according to the third aspect of the inventioncauses an inhibition of cell proliferation of the contacted cells. Thephrase “inhibiting cell proliferation” is used to denote an ability ofan inhibitor of histone deacetylase to retard the growth of cellscontacted with the inhibitor as compared to cells not contacted. Anassessment of cell proliferation can be made by counting contacted andnon-contacted cells using a Coulter Cell Counter (Coulter, Miami, Fla.)or a hemacytometer. Where the cells are in a solid growth (e.g., a solidtumor or organ), such an assessment of cell proliferation can be made bymeasuring the growth with calipers and comparing the size of the growthof contacted cells with non-contacted cells.

Preferably, growth of cells contacted with the inhibitor is retarded byat least 50% as compared to growth of non-contacted cells. Morepreferably, cell proliferation is inhibited by 100% (i.e., the contactedcells do not increase in number). Most preferably, the phrase“inhibiting cell proliferation” includes a reduction in the number orsize of contacted cells, as compared to non-contacted cells. Thus, aninhibitor of histone deacetylase according to the invention thatinhibits cell proliferation in a contacted cell may induce the contactedcell to undergo growth retardation, to undergo growth arrest, to undergoprogrammed cell death (i.e., to apoptose), or to undergo necrotic celldeath.

The cell proliferation inhibiting ability of the histone deacetylaseinhibitors according to the invention makes them useful research toolsto study the role of histone deacetylase in various biologicalprocesses. For example, the cell proliferation inhibiting ability of thehistone deacetylase inhibitors according to the invention allow thesynchronization of a population of asynchronously growing cells. Forexample, the histone deacetylase inhibitors of the invention may be usedto arrest a population of non-neoplastic cells grown in vitro in the G1or G2 phase of the cell cycle. Such synchronization allows, for example,the identification of gene and/or gene products expressed during the G1or G2 phase of the cell cycle. Such synchronization of cultured cellsmay also be useful for testing the efficacy of a new transfectionprotocol, where transfection efficiency varies and is dependent upon theparticular cell cycle phase of the cell to be transfected. Use of thehistone deacetylase inhibitors of the invention allows thesynchronization of a population of cells, thereby aiding detection ofenhanced transfection efficiency.

In some preferred embodiments, the contacted cell is a neoplastic cell.The term “neoplastic cell” is used to denote a cell that shows aberrantcell growth. Preferably, the aberrant cell growth of a neoplastic cellis increased cell growth. A neoplastic cell may be a hyperplastic cell,a cell that shows a lack of contact inhibition of growth in vitro, abenign tumor cell that is incapable of metastasis in vivo, or a cancercell that is capable of metastasis in vivo and that may recur afterattempted removal. The term “tumorigenesis” is used to denote theinduction of cell proliferation that leads to the development of aneoplastic growth. In some embodiments, the histone deacetylaseinhibitor induces cell differentiation in the contacted cell. Thus, aneoplastic cell, when contacted with an inhibitor of histone deacetylasemay be induced to differentiate, resulting in the production of anon-neoplastic daughter cell that is phylogenetically more advanced thanthe contacted cell.

In some preferred embodiments, the contacted cell is in an animal. Thus,the invention provides a method for treating a cell proliferativedisease or condition in an animal, comprising administering to an animalin need of such treatment a therapeutically effective amount of ahistone deacetylase inhibitor of the invention. Preferably, the animalis a mammal, more preferably a domesticated mammal. Most preferably, theanimal is a human.

The term “cell proliferative disease or condition” is meant to refer toany condition characterized by aberrant cell growth, preferablyabnormally increased cellular proliferation. Examples of such cellproliferative diseases or conditions include, but are not limited to,cancer, restenosis, and psoriasis. In particularly preferredembodiments, the invention provides a method for inhibiting neoplasticcell proliferation in an animal comprising administering to an animalhaving at least one neoplastic cell present in its body atherapeutically effective amount of a histone deacetylase inhibitor ofthe invention.

It is contemplated that some compounds of the invention have inhibitoryactivity against a histone deacetylase from a protozoal source. Thus,the invention also provides a method for treating or preventing aprotozoal disease or infection, comprising administering to an animal inneed of such treatment a therapeutically effective amount of a histonedeacetylase inhibitor of the invention. Preferably the animal is amammal, more preferably a human. Preferably, the histone deacetylaseinhibitor used according to this embodiment of the invention inhibits aprotozoal histone deacetylase to a greater extent than it inhibitsmammalian histone deacetylases, particularly human histone deacetylases.

The present invention further provides a method for treating a fungaldisease or infection comprising administering to an animal in need ofsuch treatment a therapeutically effective amount of a histonedeacetylase inhibitor of the invention. Preferably the animal is amammal, more preferably a human. Preferably, the histone deacetylaseinhibitor used according to this embodiment of the invention inhibits afungal histone deacetylase to a greater extent than it inhibitsmammalian histone deacetylases, particularly human histone deacetylases.

The term “therapeutically effective amount” is meant to denote a dosagesufficient to cause inhibition of histone deacetylase activity in thecells of the subject, or a dosage sufficient to inhibit cellproliferation or to induce cell differentiation in the subject.Administration may be by any route, including, without limitation,parenteral, oral, sublingual, transdermal, topical, intranasal,intratracheal, or intrarectal. In certain particularly preferredembodiments, compounds of the invention are administered intravenouslyin a hospital setting. In certain other preferred embodiments,administration may preferably be by the oral route.

When administered systemically, the histone deacetylase inhibitor ispreferably administered at a sufficient dosage to attain a blood levelof the inhibitor from about 0.01 μM to about 100 μM, more preferablyfrom about 0.05 μM to about 50 μM, still more preferably from about 0.1μM to about 25 μM, and still yet more preferably from about 0.5 μM toabout 25 μM. For localized administration, much lower concentrationsthan this may be effective, and much higher concentrations may betolerated. One of skill in the art will appreciate that the dosage ofhistone deacetylase inhibitor necessary to produce a therapeutic effectmay vary considerably depending on the tissue, organ, or the particularanimal or patient to be treated.

In certain preferred embodiments of the third aspect of the invention,the method further comprises contacting the cell with an antisenseoligonucleotide that inhibits the expression of a histone deacetylase.The combined use of a nucleic acid level inhibitor (e.g., antisenseoligonucleotide) and a protein level inhibitor (i.e., inhibitor ofhistone deacetylase enzyme actvity) results in an improved inhibitoryeffect, thereby reducing the amounts of the inhibitors required toobtain a given inhibitory effect as compared to the amounts necessarywhen either is used individually. The antisense oligonucleotidesaccording to this aspect of the invention are complementary to regionsof RNA or double-stranded DNA that encode HDAC-1, HDAC-2, HDAC-3,HDAC-4, HDAC-5, HDAC-6, HDAC7, and/or HDAC-8 (see e.g., GenBankAccession Number U50079 for HDAC-1, GenBank Accession Number U31814 forHDAC-2, and GenBank Accession Number U75697 for HDAC-3).

For purposes of the invention, the term “oligonucleotide” includespolymers of two or more deoxyribonucleosides, ribonucleosides, or2′-substituted ribonucleoside residues, or any combination thereof.Preferably, such oligonucleotides have from about 6 to about 100nucleoside residues, more preferably from about 8 to about 50 nucleosideresidues, and most preferably from about 12 to about 30 nucleosideresidues. The nucleoside residues may be coupled to each other by any ofthe numerous known internucleoside linkages. Such internucleosidelinkages include without limitation phosphorothioate,phosphorodithioate, alkylphosphonate, alkylphosphonothioate,phosphotriester, phosphoramidate, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridged phosphorothioateand sulfone internucleoside linkages. In certain preferred embodiments,these internucleoside linkages may be phosphodiester, phosphotriester,phosphorothioate, or phosphoramidate linkages, or combinations thereof.The term oligonucleotide also encompasses such polymers havingchemically modified bases or sugars and/ or having additionalsubstituents, including without limitation lipophilic groups,intercalating agents, diamines and adamantane.

For purposes of the invention the term “2′-substituted ribonucleoside”includes ribonucleosides in which the hydroxyl group at the 2′ positionof the pentose moiety is substituted to produce a 2′-O-substitutedribonucleoside. Preferably, such substitution is with a lower alkylgroup containing 1-6 saturated or unsaturated carbon atoms, or with anaryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl orallyl group may be unsubstituted or may be substituted, e.g., with halo,hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carbalkoxyl, or amino groups. The term “2′-subsbtuted ribonucleoside”also includes ribonucleosides in which the 2′-hydroxyl group is replacedwith an amino group or with a halo group, preferably fluoro.

Particularly preferred antisense oligonucleotides utilized in thisaspect of the invention include chimeric oligonucleotides and hybridoligonucleobdes.

For purposes of the invention, a “chimeric oligonucleotide” refers to anoligonucleotide having more than one type of internucleoside linkage.One preferred example of such a chimeric oligonucleotide is a chimericoligonucleotide comprising a phosphorothioate, phosphodiester orphosphorodithioate region, preferably comprising from about 2 to about12 nucleotides, and an alkylphosphonate or alkylphosphonothioate region(see e.g., Pederson et al. U.S. Pat. Nos. 5,635,377 and 5,366,878).Preferably, such chimeric oligonucleotides contain at least threeconsecutive internucleoside linkages selected from phosphodiester andphosphorothioate linkages, or combinations thereof.

For purposes of the invention, a “hybrid oligonucleotide” refers to anoligonucleotide having more than one type of nucleoside. One preferredexample of such a hybrid oligonucleotide comprises a ribonucleotide or2′-substituted ribonucleotide region, preferably comprising from about 2to about 12 2′-substituted nucleotides, and a deoxyribonucleotideregion. Preferably, such a hybrid oligonucleotide contains at leastthree consecutive deoxyribonucleosides and also containsribonucleosides, 2′-substituted ribonucleosides, preferably2′-O-substituted ribonucleosides, or combinations thereof (see e.g.,Metelev and Agrawal, U.S. Pat. No. 5,652,355).

The exact nucleotide sequence and chemical structure of an antisenseoligonucleotide utilized in the invention can be varied, so long as theoligonucleotide retains its ability to inhibit expression of the gene ofinterest. This is readily determined by testing whether the particularantisense oligonucleotide is active. Useful assays for this purposeinclude quantitating the mRNA encoding a product of the gene, a Westernblotting analysis assay for the product of the gene, an activity assayfor an enzymatically active gene product, or a soft agar growth assay,or a reporter gene construct assay, or an in vivo tumor growth assay,all of which are described in detail in this specification or inRamchandani et al. (1997) Proc. Natl. Acad. Sci. USA 94: 684-689.

Antisense oligonucleotides utilized in the invention may conveniently besynthesized on a suitable solid support using well known chemicalapproaches, including H-phosphonate chemistry, phosphoramiditechemistry, or a combination of H-phosphonate chemistry andphosphoramidite chemistry (i.e., H-phosphonate chemistry for some cyclesand phosphoramidite chemistry for other cycles). Suitable solid supportsinclude any of the standard solid supports used for solid phaseoligonucleotide synthesis, such as controlled-pore glass (CPG) (see,e.g., Pon, R. T. (1993) Methods in Molec. Biol. 20: 465-496).

Particularly preferred oligonucleotides have nucleotide sequences offrom about 13 to about 35 nucleotides which include the nucleotidesequences shown in Table 1. Yet additional particularly preferredoligonucleotides have nucleotide sequences of from about 15 to about 26nucleotides of the nucleotide sequences shown in Table 1. TABLE 1position Accession Nucleotide within Oligo Target Number PositionSequence Gene HDAC1 AS1 Human HDAC1 U50079 1585-16045′-GAAACGTGAGGGACTCAGCA-3′ 3′-UTR HDAC1 AS2 Human HDAC1 U50079 1565-15845′-GGAAGCCAGAGCTGGAGAGG-3′ 3′-UTR HDAC1 MM Human HDAC1 U50079 1585-16045′-GTTAGGTGAGGCACTGAGGA-3′ 3′-UTR HDAC2 AS Human HDAC2 U31814 1643-16225′-GCTGAGCTGTTCTGATTTGG-3′ 3′-UTR HDAC2 MM Human HDAC2 U31814 1643-16225′-CGTGAGCACTTCTCATTTCC-3′ 3′-UTR HDAC3 AS Human HDAC3 AF0397031276-1295 5′-CGCTTTCCTTGTCATTGACA-3′ 3′-UTR HDAC3 MM Human HDAC3AF039703 1276-1295 5′-GCCTTTCCTACTCATTGTGT-3′ 3′-UTR HDAC4 AS1 HumanHDAC4 AB006626 514-33 5-GCTGCCTGCCGTGCCCACCC-3′ 5′-UTR HDAC4 MM1 HumanHDAC4 AB006626 514-33 5′-CGTGCCTGCGCTGCCCACGG-3′ 5′-UTR HDAC4 AS2 HumanHDAC4 AB006626 7710-29 5′-TACAGTCCATGCAACCTCCA-3′ 3′-UTR HDAC4 MM4 HumanHDAC4 AB006626 7710-29 5′-ATCAGTCCAACCAACCTCGT-3′ 3′-UTR HDAC5 AS HumanHDAC5 AF039691 2663-2682 5′-CTTCGGTCTCACCTGCTTGG-3′ 3′-UTR HDAC6 ASHuman HDAC6 AJ011972 3791-3810 5′-CAGGCTGGAATGAGCTACAG-3′ 3′-UTR HDAC6MM Human HDAC6 AJ011972 3791-3810 5′-GACGCTGCAATCAGGTAGAC-3′ 3′-UTRHDAC7 AS Human HDAC7 AF239243 2896-2915 5′-CTTCAGCCAGGATGCCCACA-3′3′-UTR HDAC8 AS1 Human HDAC8 AF230097 51-70 5′-CTCCGGCTCCTCCATCTTCC-3′5′-UTR HDAC8 AS2 Human HDAC8 AF230097 1328-13475′-AGCCAGCTGCCACTTGATGC-3′ 3′-UTR

The following examples are intended to further illustrate certainpreferred embodiments of the invention, and are not intended to limitthe scope of the invention.

EXAMPLES Example 1N-(2-Amino-phenyl)-4-[3-(3,4-dichloro-phenyl)-acryloyl]-benzamide (Ia)

Step 1: 4-[3-(3,4-dichloro-phenyl)-acryloyl]-benzoic acid (IIa)

To a stirred suspension at room temperature of 4-acetylbenzoic acid(1.71 g, 10.44 mmol), 3,4-dichlorobenzaldehyde (2.05 g, 11.49 mmol) oraldehyde (1.1 equiv.) in MeOH (50 ml) was added a solution of NaOH (26.1ml, 1N in H₂O). After 19 h, the reaction mixture was filtered off,rinsed with MeOH and dried to afford the title compound IIa (3.22 g,10.03 mmol, 96% yield) as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆)δ(ppm): 8.35 (s, 1H), AB system (δ_(A)=8.13, δ_(B)=8.01, J=8.4 Hz, 4H),8.12 (d, J=15.8 Hz, 1H), 7.93 (d, J=7.9 Hz, 1H), 7.76 (d, J=8.3 Hz, 1H,7.73 (d, 15.8 Hz, 1H).

Step 2:N-(2-Amino-phenyl)-4-[3-(3,4-dichloro-phenyl)-acryloyl]-benzamide (Ia)

To a stirred solution at room temperature of IIa (300 mg, 0.93 mmol) inanhydrous DMF (15 ml) under nitrogen were added Et₃N (156 μl, 1.12 mmol)and BOP reagent (454 mg, 1.03 mmol), respectively. After 30 min, asolution of 1,2-phenylenediamine (111 mg, 1.03 mmol), Et₃N (391 μl, 2.80mmol) in anhydrous DMF (2 ml) was added dropwise. After 21 h, thereaction mixture was poured into a saturated aqueous solution of NH₄Cl,and diluted with AcOEt. After separation, the organic layer wassuccessively washed with sat NH₄Cl, H₂O and brine, and concentrated. Thecrude residue was then purified by flash chromatography on silica gel(AcOEt/CH₂Cl₂:10/90→20/90) to afford the title compound Ia (237 mg, 0.58mmol, 62% yield) as a yellow powder. ¹H NMR (300 MHz, DMSO-d₆) δ(ppm):9.90 (s, 1H), 8.40-8.30 (m, 3H), 8.25-8.10 (m, 3H), 7.97 (d, J=8.8 Hz,1H), 7.85-7.75 (m, 2H), 7.23 (d, J=7.5 Hz, 1H), 7.03 (t, J=7.3 Hz, 1H),6.84 (d, J=7.9 Hz, 1H), 6.65 (t, J=7.5 Hz, 1H), 4.99 (s, 2H).

Examples 2 and 10

Examples 2 and 10 (compounds Ib,Ij) were prepared using the sameprocedure as described for compound Ia of Example 1 (Scheme 1).

Example 3N-(2-Amino-phenyl)-4-[3-(2,6-dichloro-phenyl)-acryloyl]benzamide (Ic)

Step 1: t-Butyl [2-(4-acetyl-benzoylamino)-phenyl]-carbamate (III)

To a stirred solution at room temperature of 4-acetylbenzoic acid (395mg, 2.41 mmol) in anhydrous DMF (15 ml) under nitrogen were added Et₃N(369 μl, 2.65 mmol) and BOP reagent (1.171 g, 2.65 mmol), respectively.After 30 min, a solution of t-butyl (2-amino-phenyl)-carbamate (551 mg,2.65 mmol), Et₃N (1.01 ml, 7.22 mmol) in anhydrous DMF (5 ml) was addeddropwise. After 19 h, the reaction mixture was poured into a saturatedaqueous solution of NH₄Cl, and diluted with AcOEt. After separation, theorganic layer was successively washed with sat NH₄Cl, H₂O and brine,dried over anhydrous MgSO₄, filtered and concentrated. The crude residuewas then purified by flash chromatography on silica gel(AcOEt/hexane:40/60→50/50) to afford the title compound III (500 mg,1.41 mmol, 59% yield) as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ(ppm):9.47 (bs, 1H), 8.10-8.00 (m, 4H), 7.91 (d, J=7.9 Hz, 1H), 7.33-7.15 (m,3H), 6.67 (s, 1H), 2.67 (s, 3H), 1.53 (s, 9H).

Step 2: t-Butyl(2-{4-[3-(2,6-dichloro-phenyl)-acryloyl]-benzoylamino}-phenyl)-carbamate(IVc)

To a stirred solution at room temperature of III (150 mg, 0.42 mmol),2,6-dichlorobenzaldehyde (148 mg, 0.85 mmol) or aldehyde (1.5-2.0equiv.) in MeOH (10 ml) was added a solution of NaOH (1.7 ml, 1N inH₂O). A pale yellow precipitate appeared. After 3 days, the reactionmixture was filtered off, rinsed with H₂O. The solid residue was thendissolved in AcOEt, dried over anhydrous MgSO₄, filtered andconcentrated. The crude residue was finally purified by flashchromatography on silica gel (AcOEt/hexane:20/80→40/60) to afford thetitle compound IVc (185 mg, 0.36 mmol, 85% yield) as a pale yellow foam.¹H NMR (300 MHz, CDCl₃) δ(ppm): 9.48 (bs, 1H), 8.11 (s, 4H), 7.96 (d,J=17.1 Hz, 1H), 7.89 (d, J=8.8 Hz, 1H), 7.68 (d, J=16.3 Hz, 1H), 7.42(d, J=7.9 Hz, 2H), 7.35-7.15 (m, 4H), 6.68 (s, 1H), 1.54 (s, 9H).

Step 3:N-(2-Amino-phenyl)-4-[3-(2,6-dichloro-phenyl)-acryloyl]-benzamide (Ic)

To a stirred solution at room temperature of IVc (135 mg, 0.26 mmol) inCH₂Cl₂ (10 ml) was added trifluoroacetic acid (2 ml, 95% in water).After 16 h, the reaction mixture was concentrated, and directly purifiedby flash chromatography on silica gel (AcOEt/CH₂Cl₂:15/85) to afford thetitle compound Ic (90 mg, 0.22 mmol, 83% yield) as an orange solid. ¹HNMR (300 MHz, DMSO-d₆) δ(ppm): 9.89 (s, 1H), AB system (δ_(A)=8.23,δ_(B)=8.19, J=8.5 Hz, 4H), 7.90 (d, J=16.3 Hz, 1H), 7.78 (d, J=16.3 Hz,1H), 7.67 (d, J=7.9 Hz, 2H), 7.55-7.45 (m, 1H), 7.23 (d, J=7.5 Hz, 1H),7.03 (t, J=7.5 Hz, 1H), 6.83 (d, J=7.9 Hz, 1H), 6.64 (t, J=7.3 Hz, 1H),5.00 (s, 2H).

Examples 4-9

Examples 4 to 9 (compounds Id-Ii) were prepared using the same procedureas described for compound Ic of Example 3 (Scheme 2). TABLE 2 I

Cmpd Ar Name Characterization Scheme Ib

N-(2-Amino-phenyl)-4-(3- pyridin-3-yl-acryloyl)- benzamide ¹H NMR (300MHz, DMSO-d₆) δ (ppm) 9.89 (s, 1H), 9.10 (s, 1H), 8.68 (d, J = 4.8 Hz,1H), 8.44 (d, J = 7.9 Hz, 1H), AB system (δ_(A) =8.34, δ_(B) = 8.20, J =8.1 Hz, 4H), 8.19 (d, J = 15.8 Hz, 1H), 7.87 (d, J = 15.8 Hz, 1H),7.60-7.50 (m, 1H), 7.23 (d, J = 7.9 Hz, 1H), 7.04 (t, J = 7.0 #Hz, 1H),6.84 (d, J = 7.9 Hz, 1H), 6.65 (t, J = 7.3 Hz, 1H), 4.99 (s, 2H). 1 Id

N-(2-Amino-phenyl)-4-[3- (3,4,5-trimethoxy- phenyl)-acryloyl]- benzamide¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 9.89 (s, 1H), AB system (δ_(A) =8.31,δ_(B) = 8.20, J = 8.4 Hz, 4H), 7.98 (d, J = 15.8 Hz, 1H), 7.78 (d, J=15.4 Hz, 1H), 7.31 (s, 2H), 7.23 (d, J = 7.9 Hz, 1H), 7.04 (t, J =7.0Hz, 1H), 6.84 (d, J = 7.5 Hz, 1H), 6.65 (t, J = 7.5 # Hz, 1H), 4.99 (bs,2H), 3.92 (s, 6H), 3.77 (s, 3H). 2 Ie

N-(2-Amino-phenyl)-4-[3- (4-chloro-phenyl)- acryloyl]-benzamide ¹H NMR(300 MHz, DMSO-d₆) δ (ppm) 9.90 (s, 1H), AB system (δ_(A) =8.33, δ_(B) =8.19, J = 8.4 Hz, 4H), 8.08 (d, J = 15.4 Hz, 1H), AB system (δ_(A) =8.02, δ_(B) = 7.60, J = 8.6 Hz, 4H), 7.83 (d, J = 15.4 Hz, 1H), 7.22 (d,J = 7.5 Hz, 1H), 7.03 (t, J = #7.5 Hz, 1H), 6.83 (d, J = 7.9 Hz, 1H),6.65 (t, J = 7.5 Hz, 1H), 5.00 (bs, 2H). 2 If

N-(2-Amino-phenyl)-4-[3- (2,4,6-trimethoxy- phenyl)-acryloyl]- benzamide¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 9.86 (bs, 1H), 8.26-8.03 (m, 5H), 7.92(d, J = 15.8 Hz, 1H), 7.23 (d, J = 7.5 Hz, 1H), 7.03 (t, J =7.5 Hz, 1H),6.83 (d, J = 7.5 Hz, 1H), 6.65 (t, J = 6.8 Hz, 1H), 6.38 (s, 2H), 4.99(bs, 2H), 3.98 (s, 6H), 3.91 (s, 3H). 2 Ig

N-(2-Amino-phenyl)-4-[3- (3-cyclopropoxy-4- difluoromethoxy-phenyl)-acryloyl]-benzamide ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 9.89 (s, 1H), ABsystem (δ_(A) =8.31, δ_(B) = 8.20, J = 8.1 Hz, 4H), 8.00 (d, J = 15.8Hz, 1H), 7.83 (d, J = 15.4 Hz, 1H), ABX system (δ_(A) = 7.30, δ_(B) =7.64, δ_(X) = 7.96, J = 8.4, #1.3, 0 Hz, 3H), 7.23 (d, J = 7.5 Hz, 1H),7.17 (t, J = 74.3 Hz, 1H), 7.04 (t, J = 7.3 Hz, 1H), 6.84 (d, J = 7.5Hz, 1H), 6.65 (t, J =7.3 Hz, 1H), 5.00 (bs, 2H), 4.15-4.08 (m, 1H),0.98-0.73 (m, 4H). 2 Ih

N-(2-Amino-phenyl)-4-[3- (4-trifluoromethyl- phenyl)-acryloyl]-benzamide ¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 9.91 (bs, 1H), 8.35 (d, J =7.9 Hz, 2H), 8.30-8.10 (m, 5H), 7.95-7.82 (m, 3H), 7.23 (d, J = 7.9 Hz,1H), 7.04 (t, J = 7.3 Hz, 1H), 6.83 (d, J = 7.5 Hz, 1H), 6.65 (t, J =7.3 Hz, 1H), 5.00 (bs, 2H). 2 Ii

N-(2-Amino-phenyl)-4-[3- (2,5-difluoro-phenyl)- acryloyl]-benzamide ¹HNMR (300 MHz, DMSO-d₆) δ (ppm) 9.90 (s, 1H), 8.34 (d, J = 7.9 Hz, 2H),8.25-8.05 (m, 4H), 7.86 (d, J = 15.8 Hz, 1H), 751-7.38 (m, 2H), 7.23 (d,J = 7.5 Hz, 1H), 7.04 (t, J = 7.3 Hz, 1H), 6.84 (d, J = 7.9 Hz, 1H),6.65 (t, J = 7.3 Hz, 1H), 5.00 (bs, 2H). 2 Ij

N-(2-Amino-phenyl)-4-[3- (3-cyclopentyloxy-4- methoxy-phenyl)-acryloyl]-benzamide ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 9.88 (s, 1H), ABsystem (δ_(A) = 8.29, δ_(B) = 8.19, J = 7.9 Hz, 4H), 7.88 (d, J = 15.8Hz, 1H), 7.78 (d, J = 15.8 Hz, 1H), 7.55 (s, 1H), 7.49 (d, J = 8.4 Hz,1H), 7.23 (d, J = 7.5 Hz, 1H), 7.08 (d, J = 8.4 Hz, # 1H), 7.04 (t, J =7.5 Hz, 1H), 6.84 (d, J = 7.5 Hz, 1H), 6.65 (t, J = 7.3 Hz, 1H), 4.99(bs, 3H), 3.86 (s, 3H), 2.10-1.55 (m, 8H). 1

Example 11N-(2-Amino-phenyl)-4-[2-(3,4,5-trimethoxy-phenylcarbamoyl)-vinyl]-benzamide(Va)

Step 1: Methyl 4-(2-t-butoxvcarbonyl-vinyl)-benzoate (VI)

To a solution of anhydrous i-Pr₂NH (1.76 ml, 12.49 mmol) in anhydrousTHF (30 ml) stirred at 0° C. under nitrogen, was slowly added a solutionof n-BuLi (5.36 ml, 13.40 mmol, 2.5 M in hexane). After 30 min, LDA wascooled to −78° C. and t-butyl acetate (1.64 ml, 12.18 mmol) was addeddropewise. After 30 min, a solution of methyl 4-formylbenzoate (1.00 g,6.09 mmol) in anhydrous THF (10 ml) was slowly added. After 2 h, asolution of 2-chloro-4,6-dimethoxy-1,3,5-triazine (1.604 g, 9.14 mmol)in anhydrous THF (10 ml) was added. Then, the temperature was allowed towarm up to room temperature overnight. A suspension appeared. Thereaction mixture was poured into a saturated aqueous solution of NH₄Cl,and diluted with AcOEt. After separation, the organic layer wassuccessively washed with H₂O and brine, dried over MgSO₄, filtered andconcentrated. The crude product was purified by flash chromatography onsilica gel (AcOEt/hexane:10/90→15/85) to give the title product VI (785mg, 3.00 mmol, 49% yield) as a white solid. ¹H NMR (300 MHz, CDCl₃)δ(ppm): AB system (δ_(A)=8.04, δ_(B)=7.57, J=8.4 Hz, 4H), 7.60 (d,J=15.4 Hz, 1H), 6.46 (d, J=15.8 Hz, 1H), 3.93 (s, 3H), 1.54 (s, 9H). ¹³CNMR (75 MHz, CDCl₃) δ(ppm): 166.72, 166.01, 142.31, 139.18, 131.33,130.26, 127.99, 122.87, 81.11, 52.46, 28.40.

Step 2: Methyl 4-(2-carboxy-vinyl)-benzoate (VII)

To a stirred solution at room temperature of VI (745 mg, 2.84 mmol) inCH₂Cl₂ (10 ml) was added trifluoroacetc acid (6 ml, 95% in water). After27 h, the reaction mixture was concentrated, and triturated in water.After 1 h, the suspension was filtered off, rinsed with H₂O, and driedto afford the title compound VII (556 mg, 2.70 mmol, 95% yield) as anoff-white solid. ¹H NMR (300 MHz, DMSO-d₆) δ(ppm): AB system(δ_(A)=8.01, δ_(B)=7.88, J=8.1 Hz, 4H), 7.68 (d, J=15.8 Hz, 1H), 6.70(d, J=16.3 Hz, 1H), 3.90 (s, 3H).

Method A. Step 3: Methyl4-[2-(benzotriazol-1-yloxycarbonyl)-vinyl]-benzoate (VIII)

To a stirred solution at room temperature of VII (264 mg, 1.28 mmol) inanhydrous DMF (10 ml) under nitrogen were added Et₃N (196 μl, 1.41 mmol)and BOP reagent (680 mg, 1.1.54 mmol), respectively. After few min, aprecipitate appeared. After 3 h, the reaction mixture was poured into asaturated aqueous solution of NH₄Cl, and diluted with AcOEt. Afterseparation, the organic layer was successively washed with sat NH₄Cl,H₂O and brine, concentrated a little bit, and hexane was added. Thesuspension was filtered off and rinsed with hexane. The solid wastriturated in water, filtered off, rinsed with water, and dried toafford the title compound VIII (346 mg, 1.07 mmol, 84% yield) as a paleyellow solid (not stable on silica gel !). ¹H NMR (300 MHz, CDCl₃)δ(ppm): 8.56 (d, J=8.3 Hz, 1H), 8.21-8.02 (m, 3H), 7.90-7.72 (m, 4H),7.62 (t, J=7.4 Hz, 1H), 3.97 (s, 3H).

Step 4: Methyl 4-[2-(3,4,5-trimethoxy-phenylcarbamoyl)-vinyl]-benzoate(IXa)

To a stirred suspension at room temperature of VIII (150 mg, 0.46 mmol)in anhydrous CH₂Cl₂ (10 ml) under nitrogen were added Et₃N (194 μl, 1.39mmol) and 3,4,5-trimethoxyaniline (94 mg, 0.51 mmol) or ArNH₂(1.1-1.2equiv.), respectively. The reaction mixture was heated to 60° C. After20 h, the reaction mixture was concentrated, diluted with AcOEt, andsuccessively washed with a saturated aqueous solution of NH₄Cl, H₂O andbrine, dried over MgSO₄, filtered and concentrated. The crude productwas purified by flash chromatography on silica gel(AcOEt/CH₂Cl₂:15/85→20/80) to give the title product IXa (130 mg, 0.35mmol, 75% yield) as a yellow solid. ¹H NMR (300 MHz, acetone-d₆) δ(ppm):9.42 (bs, 1H), AB system (δ_(A)=8.09, δ_(B)=7.78, J=8.1 Hz, 4H), 7.75(d, J=15.6 Hz, 1H), 7.21 (s, 2H), 7.00 (d, J=15.8 Hz, 1H), 3.94 (s, 3H),3.85 (s, 6H), 3.73 (s, 3H).

Step 5: 4-[2-(3,4,5-Trimethoxy-phenylcarbamoyl)-vinyl]-benzoate (Xa)

To a stirred solution at room temperature of IXa (125 mg, 0.34 mmol) inTHF (5 ml) was added a solution of LiOH.H₂O (35 mg, 0.84 mmol) in water(5 ml). After 1.5 day, the reaction mixture was concentrated, dilutedwith water and acidified with 1N HCl until pH 4-5 in order to get aprecipitate. After stirring for 10 min, the suspension was filtered off,rinsed with water, and dried to afford the title compound Xa (110 mg,0.31 mmol, 91% yield) as a pale yellow solid. ¹H NMR (300 MHz, DMSO-d₆)δ(ppm): 10.29 (s, 1H), AB system (δ_(A)=8.04, δ_(B)=7.76, J=8.4 Hz, 4H),7.65 (d, J=15.8 Hz, 1H), 7.13 (s, 2H), 6.94 (d, J=15.8 Hz, 1H), 3.81 (s,6H), 3.67 (s, 3H).

Step 6:N-(2-Amino-phenyl)-4-[2-(3,4,5-trimethoxy-phenylcarbamoyl)-vinyl]-benzamide(Va)

To a stirred solution at room temperature of Xa (110 mg, 0.31 mmol) inanhydrous DMF (3 ml) under nitrogen were added Et₃N (47 μl, 0.34 mmol)and BOP reagent (163 mg, 0.37 mmol), respectively. After 30 min, asolution of 1,2-phenylenediamine (37 mg, 0.34 mmol), Et₃N (129 μl, 0.92mmol) in anhydrous DMF (1 ml) was added dropwise. After 3 h, thereaction mixture was poured into a saturated aqueous solution of NH₄Cl,and diluted with AcOEt. After separation, the organic layer wassuccessively washed with sat NH₄Cl, H₂O and brine, dried over MgSO₄,filtered, and concentrated. The crude residue was then purified by flashchromatography on silica gel (AcOEt/CH₂Cl₂:50/50→80/20) to afford thetitle compound Va (98 mg, 0.22 mmol, 71% yield) as a yellow solid. ¹HNMR (300 MHz, DMSO-d₆) δ(ppm): 10.27 (s, 1H), 9.76 (s, 1H), AB system(δ_(A)=8.09, δ_(B)=7.78, J=7.9 Hz, 4H), 7.71 (d, J=15.8 Hz, 1H), 7.22(d, J=7.5 Hz, 1H), 7.14 (s, 2H), 7.02 (t, J=7.0 Hz, 1H), 6.95 (d, J=15.8Hz, 1H), 6.83 (d, J=7.9 Hz, 1H), 6.65 (t, J=7.5 Hz, 1H), 4.97 (bs, 2H),3.81 (s, 6H), 3.68 (s, 3H).

Example 12N-(2-Amino-phenyl)-4-{2-[(pyridin-3-ylmethyl)-carbamoyl]-vinyl}-benzamide(Vb)

Method B, Step 3: Methyl4-[2-(pyridin-3-ylmethyl)-carbamoyl)-vinyl]-benzoate (Vb)

To a stirred solution at room temperature of VIII (140 mg, 0.68 mmol) inanhydrous DMF (5 ml) under nitrogen were added Et₃N (104 μl, 0.75 mmol)and BOP reagent (331 mg, 0.75 mmol), respectively. After 30 min, asolution of 3-(aminomethyl)pyridine (90 μl, 0.88 mmol) or R¹R²NH(1.2-1.3 equiv.), Et₃N (284 μl, 2.04 mmol) in anhydrous DMF (2 ml) wasadded dropwise. After 4 h, the reaction mixture was poured into asaturated aqueous solution of NH₄Cl, and diluted with AcOEt. Afterseparation, the organic layer was successively washed with sat NH₄Cl,H₂O and brine, dried over MgSO₄, filtered, and concentrated. The cruderesidue was then purified by flash chromatography on silica gel(MeOH/CH₂Cl₂:5/95→7/93) to afford the title compound IXb (185 mg, 0.62mmol, 92% yield) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ(ppm):8.67-8.44 (m, 2H), AB system (δ_(A)=8.03, δ_(B)=7.55, J=8.4 Hz, 4H),7.78-7.64 (m, 2H), 7.33-7.26 (m, 1H), 6.54 (d, J=15.8 Hz, 1H), 6.38 (bs,1H), 4.61 (d, J=6.2 Hz, 2H), 3.92 (s, 3H).

Step 4:N-(2-Amino-phenyl)-4-{2-[(pyridin-3-ylmethyl)-carbamoyl]-vinyl}-benzamide(Vb)

The title compound Vb was obtained from IXb in two steps following thesame procedure as Example 10, steps 5 and 6 (Scheme 3). ¹H NMR (300 MHz,DMSO-d₆) δ(ppm): 9.74 (s, 1H), 8.79 (t, J=5.7 Hz, 1H), 8.58 (s, 1H),8.52 (d, J=4.0 Hz, 1H), 8.06 (d, J=7.9 Hz, 2H), 7.83-7.68 (m, 3H), 7.59(d, J=15.8 Hz, 1H), 7.41 (dd, J=7.9, 4.7 Hz, 1H), 7.21 (d, J=7.9 Hz,1H), 7.02 (t, J=7.0 Hz, 1H), 6.83 (d, J=15.8 Hz, 1H), 6.82 (d, J=7.5 Hz,1H), 6.64 (t, J=7.3 Hz, 1H, 4.96 (bs, 2H), 4.48 (d, J=5.7 Hz, 2H).

Examples 13-15

Examples 13 to 15 (compounds Vc-Ve) were prepared using the sameprocedure as described for compound Vb of Example 12 (Scheme 3).

Example 16N-(2-Amino-phenyl)-4-[2-(2-pyridin-3-yl-ethylcarbamoyl)-vinyl]-benzamide(Vf)

Step 1: t-Butyl [2-(4-formyl-benzoylamino)-phenyl]-carbamate (XI)

To a stirred suspension at room temperature of 4-carboxybenzaldehyde(3.00 g, 19.98 mmol) in anhydrous CH₂Cl₂ (10 ml) under nitrogen wereadded thionyl chloride (2.19 ml, 29.97 mmol) and anhydrous DMF (387 μl,5.00 mmol), respectively. The reaction mixture was refluxed for 5 h.Then, the reaction mixture was allowed to cool to room temperature,concentrated, and diluted with anhydrous CH₂Cl₂ (20 ml) under nitrogen.This solution was canulated into a cooled mixture at −20° C. of t-butyl(2-amino-phenyl)-carbamic ester (4.575 g, 21.98 mmol), Et₃N (8.36 ml,59.95 mmol in anhydrous CH₂Cl₂ (50 ml) under nitrogen. After 1 h, thereaction mixture was allowed to warm up to room temperature. After 1 h,it was poured into a saturated aqueous solution of NH₄Cl, and extractedwith CH₂Cl₂. The combined organic layer was successively dried overMgSO₄, filtered, and concentrated. The crude residue was then purifiedby flash chromatography on silica gel (AcOEt/hexane:30/70→40/60) toafford the title compound XI (4.80 g, 14.11 mmol, 71% yield) as a paleyellow solid. ¹H NMR (300 MHz, CDCl₃) δ(ppm): 10.11 (s, 1H), 9.58 (bs,1H), AB system (δ_(A)=8.14, δ_(B)=7.99, J=8.1 Hz, 4H), 7.89 (d, J=7.9Hz, 1H), 7.35-7.10 (m, 3H), 6.75 (s, 1H), 1.53 (s, 9H).

Step 2: Methyl3-[4-(2-t-butoxycarbonylamino-phenylcarbamoyl)-phenyl]-acrylate (XII)

A stirred suspension of compound XI (500 mg, 1.47 mmol), methyl(triphenyl-phosphoranylidene)acetate (590 mg, 1.76 mmol) in anhydroustoluene (20 ml) was heated at 90° C. under nitrogen. After 2 days, thereaction mixture was concentrated and directly purified by flashchromatography on silica gel (AcOEt/hexane:30/70→40/60) to afford thetitle compound XII (568 mg, 1.43 mmol, 97% yield) as a pale yellow foam.¹H NMR (300 MHz, CDCl₃) δ(ppm): 9.32 (bs, 1H), AB system (δ_(A)=7.99,δ_(B)=7.62, J=8.4 Hz, 4H), 7.87 (d, J=7.9 Hz, 1H), 7.73 (s, J=15.8 Hz,1H), 7.32-7.13 (m, 3H), 6.69 (bs, 1H), 6.53 (d, J=16.3 Hz, 1H), 3.83 (s,3H), 1.53 (s, 9H).

Step 3: 3-[4-(2-t-Butoxycarbonylamino-phenylcarbamoyl)-phenyl]-acrylicacid (XIII)

To a stirred solution at room temperature of compound XII (560 mg, 1.41mmol) in THF (20 ml) was added a solution of LiOH.H₂O (148 mg, 3.53mmol) in water (20 ml). After 23 h, the reaction mixture wasconcentrated, diluted with water and acidified with 1N HCl until pH 4-5in order to get a white precipitate. After stirring for 15 min, thesuspension was filtered off, rinsed with water, and dried to afford thetitle compound XIII (495 mg, 1.29 mmol, 92% yield) as a white solid. ¹HNMR (300 MHz, DMSO-d₆) δ(ppm): 9.92 (s, 1H), 8.72 (bs, 1H), AB system(δ_(A)=8.02, δ_(B)=7.90, J=7.9 Hz, 4H), 7.69 (d, J=16.3 Hz, 1H),7.62-7.53 (m, 2H), 7.30-7.13 (m, 2H), 6.72 (d, J=16.3 Hz, 1H), 1.48 (s,9H)

Step 4: t-Butyl(2-{4-[2-(2-pyridin-3-yl-ethylcarbamoyl)vinyl]-benzoylamino}-phenyl)-carbamate(XIVf)

To a stirred solution at room temperature of compound XIII (80 mg, 0.21mmol) in anhydrous DMF (3 ml) under nitrogen were added Et₃N (35 μl,0.25 mmol) and BOP reagent (102 mg, 0.23 mmol), respectively. After 30min, a solution of 3-(2-aminoethyl)pyridine (51 mg, 0.42 mmol) or RXH(1.5-2.0 equiv.), Et₃N (87 μl, 0.63 mmol) in anhydrous DMF (1 ml) wasadded dropwise. After 3-5 h, the reaction mixture was poured into asaturated aqueous solution of NH₄Cl, and diluted with AcOEt. Afterseparation, the organic layer was successively washed with sat NH₄Cl,H₂O and brine, dried over MgSO₄, filtered, and concentrated to affordthe title compound XIVf. It was used in the next step without furtherpurification.

Step 5:N-(2-Amino-phenyl)-4-[2-(2-pyridin-3-yl-ethylcarbamoyl)-vinyl]-benzamide(Vf)

To a stirred solution at room temperature of XIVf in CH₂Cl₂ (15 ml) wasadded trifluoroacetic acid (2 ml, 95% in water). After 18 h, thereaction mixture was concentrated, dissolved in water, and neutralizedwith a saturated aqueous solution of NaHCO₃ until a pH=7. A pale yellowprecipitate appeared. After few minutes, the suspension was filteredoff, rinsed with H₂O, and dried to afford the title compound Vf (69 mg,0.18 mmol, 85% yield for two steps) as a pale yellow solid. ¹H NMR (300MHz, DMSO-d₆) δ(ppm): 9.72 (s, 1H), 8.53-8.41 (m, 2H), 8.29 (t, J=5.5Hz, 1H), 8.05 (d, J=8.4 Hz, 2H), 7.80-7.63 (m, 3H), 7.51 (d, J=15.8 Hz,1H), 7.37 (dd, J=7.5, 4.8 Hz, 1H), 7.21 (d, J=7.5 Hz, 1H), 7.02 (t,J=7.5 Hz, 1H), 6.82 (d, J=7.5 Hz, 1H), 6.76 (d, J=15.8 Hz, 1H), 6.64 (t,J=7.3 Hz, 1H), 4.95 (bs, 2H), 3.51 (dd, J=6.8 Hz, 2H), 2.86 (t, J=6.8Hz, 2H).

Examples 17-26

Examples 17 to 26 (compounds Vg-Vp) were prepared using the sameprocedure as described for compound Vf of Example 16 (Scheme 4). TABLE 3V

Cmpd Rx- Name Characterization Scheme Vc

N-(2-Amino-phenyl)-4-[2- (3,4,5-trimethoxy- benzylcarbamoyl)-vinyl]-benzamide ¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 9.73 (s, 1H), 8.64 (t, J =5.7 Hz, 1H), AB system (δ_(A) = 8.06, δ_(B) = 7.74, J = 8.1 Hz, 4H),7.58 (d, J = 15.8 Hz, 1H), 7.21 (d, J = 7.5 Hz, 1H), 7.02 (t, J = 7.3Hz, 1H), 6.85 (d, J = 15.8 Hz, 1H), 6.82 (d, J = 7.0 Hz, # 1H), 6.68 (s,2H), 6.64 (t, J = 7.5 Hz, 1H), 4.95 (bs, 2H), ), 4.39 (d, J = 5.7 Hz,2H), 3.81 (s, 6H), 3.67 (s, 3H). 3 Vd

N-(2-Amino-phenyl)-4-[2- (2-phenoxy- ethylcarbamoyl)-vinyl]- benzamide¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 9.73 (s, 1H), 8.48 (t, J = 5.3 Hz,1H), AB system (δ_(A) = 8.06, δ_(B) = 7.74, J = 8.3 Hz, 4H), 7.56 (d, J= 15.8 Hz, 1H), 7.34 (t, J = 7.9 Hz, 2H), 7.21 (d, J = 7.5 Hz, 1H),7.10-9.90 (m, 4H), 6.85 (d, J = 15.4 Hz, 1H), 6.82 (d, # J = 7.0 Hz,1H), 6.64 (t, J = 7.3 Hz, 1H), 4.95 (bs, 2H), ), 4.10 (t, J = 5.3 Hz,2H), 3.62 (quadruplet, J = 5.3 Hz, 2H). 3 Ve

N-(2-Amino-phenyl)-4-(3- morpholin-4-yl-3-oxo- propenyl)-benzamide ¹HNMR (300 MHz, DMSO-d₆) δ (ppm) 9.75 (s, 1H), AB system (δ_(A) = 8.05,δ_(B) = 7.90, J = 8.1 Hz, 4H), 7.61 (d, J =15.4 Hz, 1H), 7.43 (d, J =15.4 Hz, 1H), ), 7.20 (d, J = 7.9 Hz, 1H), 7.02 (t, J = 7.0 Hz, 1H),6.82 (d, J = 7.9 Hz, 1H), 6.64 (t, J = 7.5 Hz, 1H), 4.95 (bs, 2H), #3.90-3.55 (m, 8H). 3 Vg

Pyridin-3-ylmethyl 3-[4- (2-amino- phenylcarbamoyl)- phenyl]-acrylicester ¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 9.75 (s, 1H), 8.72 (s, 1H), 8.61(bs, 1H), AB system (δ_(A) = 8.05, δ_(B) = 7.92, J =7.5 Hz, 4H, included1H), 7.82 (d, J = 16.3 Hz, 1H), 7.55-7.43 (m, 1H), 7.21 (d, J = 7.5 Hz,1H), 7.01 (t, J = 7.3 Hz, 1H), 6.88 (d, J = 15.8 Hz, # 1H), 6.82 (d, J =7.5 Hz, 1H), 6.63 (t, J = 7.0 Hz, 1H), 5.33 (s, 2H), 4.95 (bs, 2H). 4 Vh

N-(2-Amino-phenyl)-4-[2- (indan-2-ylcarbamoyl)- vinyl]-benzamide ¹H NMR(300 MHz, DMSO-d₆) δ (ppm) 9.71 (s, 1H), 8.51 (d, J = 7.0 Hz, 1H), ABsystem (δ_(A) = 8.04, δ_(B) = 7.71, J = 8.4 Hz, 4H), 7.54 (d, J = 15.8Hz, 1H), 7.36-7.14 (m, 5H), 7.01 (t, J = 7.3 Hz, 1H), 6.82 (d, J = 7.5Hz, 1H), 6.77 (d, J = 16.3 Hz, 1H), 6.63 (t, J = 7.5 # Hz, 1H), 4.94(bs, 2H), 4.71-4.57 (m, 1H), 3.28 (dd, J = 16.0, 7.3 Hz, 2H), 2.87 (dd,J = 16.3, 5.3 Hz, 2H). 4 Vi

N-(2-Amino-phenyl)-4-[2- (2-pyridin-2-yl- ethylcarbamoyl)-vinyl]-benzamide ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 9.72 (s, 1H), 8.56 (d, J =4.4 Hz, 1H), 8.28 (t, J = 5.5 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H),7.82-7.66 (m, 3H), 7.52 (d, J = 15.8 Hz, 1H), 7.40-7.15 (m, 3H), 7.02(t, J = 7.3 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 15.8 Hz,1H), 6.64 (t, # J = 7.3 Hz, 1H), 4.95 (s, 2H), 3.61 (quadruplet, J = 6.6Hz, 2H), 2.99 (t, J =7.2 Hz, 2H). 4 Vj

N-(2-Amino-phenyl)-4-{2- [2-(1H-indol-3-yl)- ethylcarbamoyl]-vinyl}-benzamide ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 10.85 (bs, 1H), 9.73 (bs,1H), 8.31 (t, J = 5.6 Hz, 1H), AB system (δ_(A) = 8.06, δ_(B) = 7.73, J= 8.0 Hz, 4H), 7.61 (d, J = 9.0 Hz, 1H), 7.55 (d, J = 15.0 Hz, 1H), 7.39(d, J = 9.0 Hz, 1H), 7.28-7.15 (m, 2H), 7.11 (t, # J = 7.5 Hz, 1H), 7.03(d, J = 9.0 Hz, 1H), 6.83 (d, J = 7.5 Hz, 1H), 6.81 (d, J = 15.0 Hz,1H), 6.64 (t, J = 7.3 Hz, 1H), 4.94 (s, 2H), 3.54 (quadruplet, J = 6.7Hz, 2H), 2.94 (t, J = 6.8 Hz, 2H). 4 Vk

N-(2-Amino-phenyl)-4-{3- [4-(3,4-dimethoxy- phenyl)-piperidin-1-yl]-3-oxo-propenyl}- benzamide ¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 9.74 (s, 1H),AB system (δ_(A) = 8.05, δ_(A) = 7.91, J = 8.1 Hz, 4H), 7.60 (d, J =15.8 Hz, 1H), 7.49 (d, J =15.4 Hz, 1H), ), 7.21 (d, J = 7.5 Hz, 1H),7.02 (t, J = 7.0 Hz, 1H), 6.95-6.75 (m, 4H), 6.64 (t, # J = 7.3 Hz, 1H),4.95 (bs, 2H), 4.70 (bd, J = 11.9 Hz, 1H), 4.50 (bd, J = 12.3 Hz, 1H),3.79 and 3.75 (2s, 6H), 3.22 (bt, J = 12.3 Hz, 1H), 2.88-2.70 (m, 2H),1.96-1.80 (m, 2H), 1.75-1.47 (m, 2H). 4 Vl

(rac)-N-(2-Amino-phenyl)- 4-[3-oxo-3-(2-pyridin-3-yl-pyrrolidin-1-yl)-propenyl]- benzamide ¹H NMR (300 MHz, DMSO-d₆) δ (ppm)mixture of rotamers, 9.75 and 9.67 (2s, 1H), 8.55-8.45 (m, 2H),8.05-6.58 (m, 12H), 5.60-5.55 and 5.25-5.20 (2m, 1H), 5.00-4.90 (m, 2H),4.15-3.65 (m, 2H), 2.50-2.28 (m, 1H), 2.10-1.75 (m, 3H). 4 Vm

N-(2-Amino-phenyl)-4-[3- oxo-3-(4-pyridin-2-yl-piperazin-1-yl)-propenyl]- benzamide ¹H NMR (300 MHz, DMSO-d₆) δ (ppm)9.75 (s, 1H), 8.18 (d, J = 3.5 Hz, 1H), AB system (δ_(A) = 8.06, δ_(B) =7.92, J = 8.1 Hz, 4H), 7.70-7.55 (m, 2H) 7.49 (d, J = 15.4 Hz, 1H),),7.21 (d, J = 7.5 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.92 (d, J = 8.8 Hz,1H), # 6.83 (d, J = 7.9 Hz, 1H), 6.72 (t, J = 6.2 Hz, 1H), 6.64 (t, J =7.5 Hz, 1H), 4.95 (bs, 2H), 4.00-3.50 (m, 8H). 4 Vn

N-(2-Amino-phenyl)-4-{2- [(2-cyano-ethyl)-pyridin-3-ylmethyl-carbamoyl]- vinyl}-benzamide ¹H NMR (300 MHz, DMSO-d₆) δ(ppm) mixture of rotamers, 9.75 and 9.72 (2s, 1H), 8.65-8.45 (m, 2H),8.15-7.80 (m, 4H), 7.78-7.62 (m, 2H), 7.55-7.35 (m, 2H), 7.21 (d, J =7.5 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.83 (d, J = 7.9 Hz, 1H), 6.64(t, J = 7.3 Hz, 1H), 5.02 # (bs, 1H), 4.95 (bs, 2H), 4.74 (bs, 1H), 3.93(bs, 1H), 3.71 (t, J = 6.2 Hz, 1H),), 2.92 (t, J = 6.2 Hz, 1H),), 2.86(t, J = 6.2 Hz, 1H). 4 Vo

N-(2-Amino-phenyl)-4-[2- (bis-pyridin-3-ylmethyl- carbamoyl)-vinyl]-benzamide ¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 9.73 (s, 1H), 8.60-8.45 (m,4H), AB system (δ_(A) = 8.03, δ_(B) = 7.87, J = 8.4 Hz, 4H), 7.80-7.63(m, 3H), 7.49 (d, J = 15.4 Hz, 1H), 7.45-7.32 (m, 2H), 7.20 (d, J = 7.5Hz, 1H), 7.01 (t, J = 7.0 Hz, 1H), 6.82 (d, J = 7.5 # Hz, 1H), 6.63 (t,J = 7.3 Hz, 1H), 4.97 and 4.95 (2s, 4H), 4.70 (s, 2H). 4 Vp

(−)-(1S,2R)-N-(2-Amino- phenyl)-4-[2-(2-hydroxy- indan-1-ylcarbamoyl)-vinyl]-benzamide ¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 9.73 (s, 1H), 8.26(d, J = 8.8 Hz, 1H), AB system (δ_(A) = 8.07, δ_(B) = 7.75, J = 8.1 Hz,4H), 7.62 (d, J = 15.4 Hz, 1H), 7.35-7.15 (m, 5H), 7.13 (d, J = 15.8 Hz,1H), 7.02 (t, J = 7.5 Hz, 1H), 6.83 (d, J = 7.0 #Hz, 1H), 6.64 (t, J =7.5 Hz, 1H), 5.39 (dd, J = 8.4, 4.8 Hz, 1H), 5.19 (d, J = 4.0 Hz, 1H),4.97 (bs, 2H), 4.58-4.46 (m, 1H), 3.14 (dd, J = 16.3, 4.8 Hz, 1H), 2.88(d, J = 15.8, 5.3 Hz, 1H). 4

Example 27N-(2-Amino-phenyl)-4-[3-(3-cyclopentyloxy-4-methoxy-phenylamino)-propenyl]-benzamide(XVa)

Step 1: t-Butyl {2-[4-(3-oxo-propenyl)-benzoylamino]-phenyl}-carbamate(XVI)

A stirred suspension of compound XI (4.00 g, 11.75 mmol),(triphenylphosphoranylidene)-acetaldehyde (3.60 g, 11.83 mmol) inanhydrous toluene (100 ml) was heated at 80° C. under nitrogen. After 2days, the reaction mixture was concentrated and directly purified byflash chromatography on silica gel (AcOEt/hexane:30/70) to afford thetitle compound XVI (3.70 g, 10.10 mmol, 86% yield) as a yellow stickysolid (slightly contaminated with the diene). ¹H NMR (300 MHz, CDCl₃)δ(ppm): 9.75 (d, J=7.8 Hz, 1H), 9.49 (bs, 1H), AB system (δ_(A)=8.03,δ_(B)=7.65, J=8.4 Hz, 4H), 7.85-7.72 (m, 1H), 7.52 (d, J=15.6 Hz, 1H),7.33-7.05 (m, 3H), 7.05-6.90 (m, 1H), 6.78 (dd, J=15.6, 7.8 Hz, 1H),1.53 (s, 9H).

Step 2: t-Butyl(2-{4-[3-(3-cyclopentyloxy-4-methoxy-phenylamino)-propenyl]-benzoylamino}-phenyl)-carbamate(XVIIa)

To a stirred solution at room temperature of compound XVI (210 mg, 0.57mmol), 3-cyclopentyloxy-4-methoxy-aniline (125 mg, 0.60 mmol) or ArNH₂(1.05-1.2 equiv.) in anhydrous THF (7 ml) under nitrogen were addeddibutyltin dichloride (3.5 mg, 0.01 mmol). After 10 min, phenylsilane(78 μl, 0.63 mmol) was added dropwise. After 3 days, the reactionmixture was concentrated and directly purified by flash chromatographyon silica gel (AcOEt/hexane:30/70→50/50) to afford the title compoundXVIIa as a yellow sticky oil.

Step 3:N-(2-Amino-phenyl)-4-[3-(3-cyclopentyloxy-4-methoxy-phenylamino)-propenyl]-benzamide(XVa)

To a stirred solution at room temperature of XVIIa in CH₂Cl₂ (30 ml) wasadded trifluoroacetic acid (5 ml, 95% in water). After 16 h, thereaction mixture was concentrated, dissolved in water, and basified witha aqueous solution of NaOH (1N) until a pH=8. A beige precipitateappeared. After 15 min, the suspension was filtered off, rinsed withH₂O, and air-dried. The crude product was purified by flashchromatography on silica gel (AcOEt/CH₂Cl₂:15/85→20/80+ε NH₄OH) toafford the title compound XVa (145 mg, 0.32 mmol, 55% yield for twosteps) as a yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ(ppm): mixture ofrotamers, 9.67 and 9.63 (2s, 1H), 7.98 (d, J=7.9 Hz, 2H), 7.57 and 7.51(2d, J=7.9 Hz, 2H), 7.20 (d, J=7.9 Hz, 1H), 7.01 (t, J=7.7 Hz, 1H), 6.82(d, J=7.9 Hz, 1H), 6.77-6.67 (m, 2H), 6.63 (t, J=7.5 Hz, 1H), 6.54 (dt,J=16.3, 5.2 Hz, 1H), 6.35 and 6.30 (2d, J=2.0 Hz, 1H), 6.15 and 6.06(2dd, J=8.6, 2.0 Hz, 1H), 5.98 and 5.57 (2t, J=5.5 Hz, 1H), 4.92 (bs,2H), 4.78-4.63 (m, 1H), 4.32 and 3.87 (2d, J=5.6 Hz, 2H), 3.65 and 3.62(2s, 3H), 1.95-1.45 (m, 8H).

Examples 28-32

Examples 28 to 32 (compounds XVb-XVf) were prepared using the sameprocedure as described for compound XVa of Example 27 (Scheme 5).

Example 33N-(2-Amino-phenyl)-4-[3-(4-tolyl-sulfonylamino)-propenyl]-benzamide(XVg)

Step 1: t-Butyl{2-[4-(3-hydroxy-propenyl)-benzoylamino]-phenyl}-carbamate (XVIII)

To a stirred solution of compound XVI (1.00 g, 2.79 mmol) in ethanol (15ml) under nitrogen was added sodium borohydride (110 mg, 2.73 mmol).After 5 min, the reaction mixture was quenched with water and dilutedwith AcOEt. After separation the organic layer was successively washedwith brine, dried over MgSO₄, filtered, and concentrated. The cruderesidue was then purified by flash chromatography on silica gel(AcOEt/hexane:40/60) to afford the title compound XVIII (910 mg, 2.29mmol, 82% yield) as a pale yellow solid (slightly contaminated with thediene). ¹H NMR (300 MHz, CDCl₃) δ(ppm): 9.20 (s, 1H), 7.90 (d, J=7.8 Hz,2H), 7.75 (d, J=7.5 Hz, 1H), 7.43 (d, J=8.4 Hz, 2H), 7.32-7.08 (m, 3H),6.94 (s, 1H), 6.65 (d, J=15.9 Hz, 1H), 6.45 (td, J=15.9, 5.4 Hz, 1H),4.35 (d, J=5.4 Hz, 2H), 1.92 (s, 1H), 1.51 (s, 9H).

Step 2: t-butyl(2-{4-[3-(4-tolyl-sulfonylamino)-propenyl]-benzoylamino}-phenyl)-carbamate(XVIIg)

To a stirred solution of N-Boc-4-tolylsulfonamide (221 mg, 0.81 mmol)and PPh₃ (427 mg, 1.63 mmol) in anhydrous THF (4 ml) under nitrogen wassuccessively added a solution of compound XVIII (200 mg, 0.54 mmol) inanhydrous THF (1 ml) and diethyl azodicarboxylate (DEAD) (214 μl, 1.36mmol). After 16 h, the reaction mixture was quenched with water anddiluted with AcOEt. After separation the organic layer was successivelywashed with water and brine, dried over MgSO₄, filtered, andconcentrated. The crude residue was then purified by flashchromatography on silica gel (AcOEt/hexane:40/60) to afford the titlecompound XVIIg (337 mg).

Step 3:N-(2-Amino-phenyl)-4-[3-(4tolyl-sulfonylamino)-propenyl]-benzamide (XVg)

To a stirred solution at room temperature of XVIIg in CH₂Cl₂ (20 ml) wasadded trifluoroacetc acid (2 ml, 95% in water). After 16 h, the reactionmixture was concentrated, dissolved in water, and basified with aaqueous saturated solution of NaHCO₃. The aqueous layer was extractedwith AcOEt. The combined organic layer was successively washed withbrine, dried over MgSO₄, filtered, and concentrated. The crude residuewas solubilized with a minimum of a mixture of AcOEt/MeOH (95/5) andcoprecipitated with hexane. An off-white precipitate appeared. After fewminutes, the suspension was filtered off, rinsed with hexane and driedto give the title compound XVg (173 mg, 0.41 mmol, 76% yield for twosteps) as an off-white solid. ¹H NMR: (300 MHz, DMSO-d₆) δ(ppm): 9.64(s, 1H), AB system (δ_(A)=7.93, δ_(B)=7.72, J=8.4 Hz, 4H), 7.84 (s, 1H),7.41 (t, J=8.4 Hz, 4H), 7.16 (d, J=7.8 Hz, 1H), 6.97 (t, J=7.5 Hz, 1H),6.77 (d, J=7.5 Hz, 1H), 6.59 (t, J=7.8 Hz, 1H), 6.52 (d, J=15.6 Hz, 1H),6.21 (dt, J=15.6, 5.7 Hz, 1H), 4.89 (s, 2H), 3.60 (bs, 2H), 2.08 (s,3H).

Example 34N-(2-Amino-phenyl)-4-{3-[(pyridin-3-ylmethyl)-amino]propenyl}-benzamide(XVh)

Step 1: Methyl 4-(3-oxo-propenyl)-benzoate (XIX)

Method A: A stirred suspension of compound methyl 4-formylbenzoate (4.00g, 24.37 mmol), (triphenylphosphoranylidene)-acetaldehyde (7.56 g, 24.85mmol) in anhydrous toluene (100 ml) was heated at 80-90° C. undernitrogen. After 1 day, the reaction mixture was concentrated anddirectly purified by flash chromatography on silica gel(AcOEt/hexane:20/80□30/70) to afford the title compound XIX (2.52 g,13.25 mmol, 54% yield) as a pale yellow solid (slightly contaminatedwith the diene). ¹H NMR (500 MHz, CDCl₃) δ(ppm): 9.76 (d, J=7.3 Hz, 1H),AB system (δ_(A)=8.11, δ=7.64, J=8.1 Hz, 4H), 7.51 (d, J=15.6 Hz, 1H),6.79 (dd, J=15.8, 7.6 Hz, 1H), 3.95 (s, 3H).

Method B: To a vigorously stirred emulsion at room temperature of TDA-1(6.278 g, 19.41 mmol) and an aqueous solution of 10% of potassiumcarbonate (100 ml) in CH₂Cl₂ (100 ml) were added(1,3-dioxolan-2-yl)methyltriphenylphosphonium bromide (10 g, 23.29 mmol)and methyl 4-formylbenzoate (3.187 g, 19.41 mmol), respectively. Afterstirring for 18 h, the reaction mixture was extracted with CH₂Cl₂ andthe combined organic layer was concentrated. Then, an aqueous solutionof 10% HCl (100 ml) was added and the mixture stirred overnight at roomtemperature. The reaction mixture was diluted with water and extractedwith CH₂Cl₂. The combined organic layer was successively dried overMgSO₄, filtered, and concentrated. The crude residue was then purifiedby flash chromatography on silica gel (AcOEt/hexane:20/80□30/70) andtriturated in AcOEt/hexane, to afford the title compound XIX (2.50 g,13.14 mmol, 68% yield) as a crystalline solid (pure trans geometry andfree of diene).

Step 2: Methyl 4-{3-[(pyridin-3-ylmethyl)-amino]-propenyl}-benzoate(XXh)

A solution at room temperature of compound XIX (300 mg, 1.58 mmol) and3-(aminomethyl)pyridine (193 μl, 0.60 mmol) or RNH₂ (1.1-1.2 equiv.) inanhydrous dichloromethane (15 ml) under nitrogen was stirred for 1 h,and sodium triacetoxyborohydride (401 mg, 1.89 mmol) was added. After 64h, the reaction mixture was quenched with an aqueous solution of K₂CO₃(10%) and extracted with dichloromethane. The combined organic layer wasdried over MgSO₄, filtered, and concentrated. The crude residue was thenpurified by flash chromatography on silica gel (MeOH/CH₂Cl₂:5/95+εNH₄OH) to afford the title compound XXh (188 mg, 0.66 mmol, 42% yield)as a dark yellow oil.

Step 3:4-[3-(tert-Butoxycarbonyl-pyridin-3-ylmethyl-amino)-propenyl]-benzoicacid (XXIh)

To a stirred solution at room temperature of XXh (187 mg, 0.66 mmol) in1,4-dioxane (7 ml) were added (Boc)₂O (173 mg, 0.80 mmol) and an aqueoussolution of NaOH (3.3 ml, 1N), respectively. After 24 h, the reactionmixture was concentrated, diluted in water, and neutralized (pH=6-7)with a aqueous solution of HCl (1N). The resulting pale yellowsuspension was extracted with dichloromethane. The combined organiclayer was dried over MgSO₄, filtered and concentrated to afford thetitle compound XXIh (160 mg, 0.43 mmol, 66% yield) as a yellow solid.

Step 4: t-Butyl{3-[4-(2-amino-phenylcarbamoyl)-phenyl]-ally}-pyridin-3-ylmethyl-carbamate(XXIIh)

The title compound XXIIh (Example 34) was obtained from XXIh aspale-yellow foam in one step following the same procedure as in Example11, step 6.

Step 5:N-(2-Amino-phenyl)-4-{3-[(pyridin-3-ylmethyl)-amino]-propenyl}-benzamide(XVh)

To a stirred solution at room temperature of XXIIh (77 mg, 0.17 mmol) indichloromethane (10 ml) was added TFA (2 ml, 95% in water). After 4.5 h,the reaction mixture was concentrated, diluted in water, basified (pH=9)with a aqueous solution of NaOH (1N), and extracted withdichloromethane. The combined organic layer was dried over MgSO₄,filtered and concentrated. The crude residue was then purified by flashchromatography on silica gel (MeOH/CH₂Cl₂:10/90+ε NH₄OH) to afford thetitle compound XVh (35 mg, 0.10 mmol, 58% yield) as a yellow powder. ¹HNMR: (400 MHz, DMSO-d₆) δ(ppm): 9.64 (s, 1H), 8.55 (s, 1H), 8.44 (d,J=3.9 Hz, 1H), AB system (δ_(A)=7.94, δ_(B)=7.55, J=8.0 Hz, 4H), 7.78(d, J=7.4 Hz, 1H), 7.36 (dd, J=7.0, 5.1 Hz, 1H), 7.16 (d, J=7.4 Hz, 1H),6.98 (t, J=7.4 Hz, 1H), 6.79 (d, J=7.8 Hz, 1H), 6.65-6.55 (m, 2H), 2.51(dt, J=16.0, 5.9 Hz, 1H), 4.93 (bs, 2H), 3.77 (s, 2H).

Examples 35-36

Examples 35 to 36 (compounds XVi-XVj) were prepared using the sameprocedure as described for compound XVh of Example 34 (Scheme 7, PathwayB). TABLE 4 XV

Cmpd Rx- Name Characterization Scheme XVb

N-(2-Amino-phenyl)-4[3- (3,4,5-trimethoxy- phenylamino)-propenyl]-benzamide ¹H NMR (300 MHz, CDCl₃) δ (ppm): 7.94 (m, 1H), AB system(δ_(A) = 7.85, δ_(B) = 7.45, J = 8.4 Hz, 4H), 7.33 (d, J = 8.1 Hz, 1H),7.09 (t, J = 7.2 Hz, 1H), 6.90-6.78 (m, 2H), 6.69 (d, J = 15.9 Hz, 1H),6.45 (td, J = 15.9, 5.7 Hz, 1H), 6.95 (s, 2H), 3.96 (d, J = 5.7 # Hz,2H), 3.82 (s, 6H), 3.77 (s, 3H). 5 XVc

N-(2-Amino-phenyl)-4-[3- (benzothiazol-2-ylamino)- propenyl]-benzamide¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 9.65 (bs, 1H), 8.30 (t, J = 4.9 Hz,1H), AB system (δ_(A) = 7.96, δ_(B) = 7.58, J = 7.8 Hz, 4H), 7.69 (d, J= 7.3 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.16(d, J = 6.8 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.97 (t, # J = 7.6 Hz,1H), 6.78 (d, J = 7.8 Hz, 1H), 6.71 (d, J = 16.1 Hz, 1H), 6.62-6.51 (m,2H), 4.89 (bs, 2H), 4.26-4.19 (m, 2H). 5 XVd

N-(2-Amino-phenyl)-4-[3- (4-methoxy-6-methyl- pyrimidin-2-ylamino)-propenyl]-benzamide ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) mixture ofrotamers, 9.63 (bs, 1H), AB system (δ_(A) = 7.93, δ_(B) = 7.53, J = 7.5Hz, 4H), 7.35-7.05 (m, 2H), 6.96 (t, J = 6.5 Hz, 1H), 6.77 (d, J =7.9Hz, 1H), 6.65-6.40 (m, 3H), 5.91 (bs, 1H), 4.15-4.03 (m, 2H), 3.80 (bs,# 3H), 2.16 (bs, 3H). 5 XVe

N-(2-Amino-phenyl)-4-[3- (5-methylsulfanyl-1H-[1,2,4]triazol-3-ylamino)- propenyl]-benzamide ¹H NMR (500 MHz, DMSO-d₆)δ (ppm): 12.16 (bs, 1H), 9.64 (s, 1H), 8.00-7.90 (m, 2H), 7.60-7.50 (m,2H), 7.20-7.13 (m, 1H), 7.02-6.85 (m, 2H), 6.82-6.75 (m, 1H), 6.67-6.55(m, 2H), 6.55-6.42 (m, 1H), 4.88 (bs, 2H), 3.93 (bs, 2H), 2.42 (s, 3H).5 XVf

4-[3-(6-Acetyl- benzo[1,3]dioxol-5- ylamino)-propenyl]-N-2-amino-phenyl)-benzamide ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 9.64 (s, 1H),9.47 (t, J = 6.0 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz,2H), 7.32 (s, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.95 (td, J = 8.4, 1.6 Hz,1H), 6.76 (dd, J = 8.4, 1.6 Hz, 1H), 6.63 (d, J = 16.0 Hz, 1H), 6.58(td, J = #8.4, 1.6 Hz, 1H), 6.54 (dt, J = 16.0, 5.6 Hz, 1H), 6.46 (s,1H), 5.97 (s, 2H), 4.88 (s, 2H), 4.07 (t, J =5.6 Hz, 2H), 2.45 (s, 3H).5 XVg

N-(2-Amino-phenyl)-4-[3- (3,4-dimethoxy- phenylamino)-propenyl]-benzamide ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 9.67 (s, 1H), AB system(δ_(A) = 7.96, δ_(B) = 7.56, J = 8.2 Hz, 4H), 7.18 (d, J = 6.7 Hz, 1H),6.99 (td, J = 7.6, 1.6 Hz, 1H), 6.80 (dd, J = 8.0, 1.4 Hz, 1H), ABXsystem (δ_(A) = 6.74, δ_(B) = 6.13, δ_(X) = 6.37, #J_(AB) = 8.5 Hz,J_(BX) = 2.6 Hz, J_(AX) = 0 Hz, 3H), 6.71 (d, J = 16.2 Hz, 1H), 6.62(td, J = 7.5, 1.4 Hz, 1H), 6.52 (dt, J = 16.0, 5.5 Hz, 1H), 5.59 (t, J =5.9 Hz, 1H), 4.92 (s, 2H), 3.88 (t, J = 4.9 Hz, 2H), 3.72 (s, 3H), 3.65(s, 3H). 7 (Pathway B) XVh

N-(2-Amino-phenyl)-4-[3- (4-pyridin-3-yl-pyrimidin-2-ylamino)-propenyl]- benzamide ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 9.65(s, 1H), 9.31 (s, 1H), 8.71 (dd, J = 4.8, 1.7 Hz, 1H), 8.53-8.42 (m,2H), 7.95 (d, J = 8.2 Hz, 2H), 7.67 (t, J = 5.9 Hz, 1H), 7.60-7.52 (m,3H), 7.30 (d, J = 5.1 Hz, 1H), 7.17 (d, J = 6.7 Hz, 1H), 6.99 (td, J=7.6, 1.6 Hz, 1H), # 6.79 (dd, J = 8.0, 1.4 Hz, 1H), 6.69 (bd, J = 16.0Hz, 1H), 6.64-6.53 (m, 2H), 4.92 (s, 2H), 4.32-4.20 (m, 2H). 7 (PathwayB)

Example 37 N-(2-Amino-phenyl)-4-(3-oxo-3-phenyl-propenyl)-benzamide(XXIVa)

Step 1: 4-(3-Oxo-3-phenyl-propenyl)-benzoic acid (XIIIa)

To a stirred suspension at room temperature of 4-formylbenzoic acid(2.58 g, 17 mmol) and acetophenone (2.0 ml, 17 mmol) or acetophenonederivatives (1.0-1.1 equiv.) in MeOH (100 ml) was added a solution ofNaOH (34 ml, 1N in H₂O). After 16 h, the reaction mixture was acidifiedwith conc. HCl (pH=1-2), filtered off, rinsed with H₂O and dried toafford the title compound XXIIIa (3.73 g, 14.6 mmol, 86% yield) as ayellow solid.

Step 2: N-(2-Amino-phenyl)-4-(3-oxo-3-phenyl-propenyl)-benzamide (XXIVa)

The title compound XXIVa was obtained from XXIIIa in one step followingthe same procedure as Example 1, step 2 (Scheme 1). ¹H NMR (300 MHz,DMSO-d₆) δ(ppm): 9.77 (s, 1H); 8.21 (d, J=7.0 Hz, 2H); 8.06 (m, 5H),7.82 (d, J=15.4 Hz, 1H), 7.71 (t, J=7.3 Hz, 1H), 7.60 (t, J=7.3 Hz, 2H),7.18 (d, J=7.9 Hz, 1H), 6.99 (t, J=7.0 Hz, 1H), 6.80 (d, J=7.5 Hz, 1H),6.61 (t, J=7.3 Hz, 1H), 4.95 (bs, 2H).

Examples 38-41

Examples 38 to 41 (compounds XXIVb-XXIVe) were prepared using the sameprocedure as described for compound XXIVa of Example 37 (Scheme 8).

Example 42N-(2-Amino-phenyl)-4-[3-(4-morpholin-4-yl-phenyl)-3-oxo-propenyl]-benzamide(XXIVf)

Step 1: t-Butyl(2-{4-[3-(4-morpholin-4-yl-phenyl)-3-oxo-propenyl]-benzoylamino}-phenyl)-carbamate

To a stirred solution at room temperature of XI (210 mg, 0.62 mmol),4′-morpholino acetophenone (227 mg, 1.11 mmol) or acetophenonederivative (1.5-2.0 equiv.) in MeOH (10 ml) was added a solution of NaOH(1.9 ml, 1N in H₂O). A precipitate appeared. After 3 days, the reactionmixture was filtered off, rinsed with MeOH, air-dried and dried undervacuum to afford the title compound XXVf (295 mg, 0.56 mmol, 90% yield)as a yellow solid.

Step 2:N-(2-Amino-phenyl)-4-[3-(4-morpholin-4-yl-phenyl)-3-oxo-propenyl]-benzamide(XXIVf)

To a stirred solution at room temperature of XXVf (285 mg, 0.54 mmol) inCH₂Cl₂ (10 ml) was added trifluoroacetic acid (2 ml, 95% in water).After 17 h, the reaction mixture was concentrated, diluted with AcOEt,successively washed with sat NaHCO₃, H₂O, sat NH₄Cl, H₂O and brine,dried over MgSO₄, filtered, and concentrated. The crude product wasco-precipitated in a mixture of AcOEt/hexane and triturated. After fewhours, the suspension was filtered off, rinsed with hexane and dried toafford the title compound XXIVf (210 mg, 0.49 mmol, 91% yield) as ayellow-orange solid. ¹H NMR (300 MHz, DMSO-d₆) δ(ppm): 9.78 (s, 1H),8.25-7.94 (m, 7H), 7.76 (d, J=15.4 Hz, 1H), 7.22 (d, J=7.5 Hz, 1H), 7.09(d, J=8.8 Hz, 2H), 7.03 (t, J=7.7 Hz, 1H), 6.83 (s, J=7.5 Hz, 1H), 6.65(t, J=7.5 Hz, 1H), 4.97 (bs, 2H), 3.88-3.70 (m, 4H), 3.48-3.30 (m, 4H).TABLE 5 XXIV

Cmpd Ar NAME Characterization Scheme XXIVb

N-(2-Amino-phenyl)-4-[3- (2-nitro-phenyl)-3-oxo- propenyl]-benzamide ¹HNMR (300 MHz, DMSO-d₆) δ (ppm) 9.73 (s, 1H), 8.22 (d, J = 7.6 Hz, 1H),8.03-7.76 (m, 7H), 7.51-7.39 (m, 2H), 7.16 (d, J = 7.6 Hz, 1H), 6.97 (t,J = 7.6 Hz, 1H), 6.78 (d, J = 7.6 Hz, 1H), 6.59 (t, J = 7.3 Hz, 1H),4.92 (bs, 2H). 8 XXIVc

N-(2-Amino-phenyl)-4-[3- (4-methoxy-phenyl)-3- oxo-propenyl]- benzamide¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 9.75 (bs, 1H), 8.21 (d, J = 8.8 Hz,2H), 8.11-8.01 (m, 5H), 7.77 (d, J = 15.4 Hz, 1H), 7.18 (d, J = 7.3 Hz,1H), 7.11 (d, J = 8.8 Hz, 2H), 6.99 (dd, J = 7.7, 7.7 Hz, 1H), 6.79 (d,J =7.3 Hz, 1H), 6.61 (dd, J = 7.3, 7.3 Hz, 1H), 4.94 (bs, 2H), 3.88 (s,3H). 8 XXIVd

N-(2-Amino-phenyl)-4-[3- (3-methoxy-phenyl)-3- oxo-propenyl]- benzamide¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 9.75 (bs, 1H), 8.08-8.03 (m, 5H),7.83-7.78 (m, 2H), 7.65 (bs, 1H), 7.51 (dd, J = 7.7, 7.7 Hz, 1H), 7.27(dd, J = 8.4, 2.3 Hz, 1H), 7.17 (d, J = 8.1 Hz, 1H), 6.98 (dd, J = 7.3,7.3 Hz, 1H), 6.79 (d, J = 7.3 Hz, 1H), 6.60 (dd, J = 7.3, 7.3 Hz, 1H),4.93 # (bs, 2H), 3.87 (s, 3H). 8 XXIVe

N-(2-Amino-phenyl)-4-[3- (2-methoxy-phenyl)-3- oxo-propenyl]- benzamide¹H NMR (300 MHz, DMSO-d₆) δ (ppm) 9.73 (bs, 1H), 8.03 (d, J = 8.1 Hz,2H), 7.88 (d, J = 8.1 Hz, 2H), 7.61-7.50 (m, 4H), 7.22 (d, J = 8.1 Hz,1H), 7.17 (d, J = 8.1 Hz, 1H), 7.08 (dd, J = 7.3, 7.3 Hz, 1H), 6.98 (dd,J = 7.7, 7.7 Hz, 1H), 6.78 (d, J = 7.3 Hz, 1H), 6.60 (dd, J = 7.7, # 7.7Hz, 1H), 4.93 (bs, 2H), 3.89 (s, 3H). 8

Example 43 N-(2-Amino-phenyl)-4-(3-oxo-3-phenyl-propyl)-benzamide(XXVIIa)

Step 1: 4-(3-Oxo-3-phenyl-propyl)-benzoic acid (XXVIa)

To a stirred solution at room temperature of chalcone XXIIIa (1.29 g,5.13 mmol) in DMF (20 ml) was added phenylsulfonylhydrazine (1.76 g,10.26 mmol). The reaction mixture was stirred at 10° C. for 15 h, cooledand concentrated. The remained oily residue was partitioned between asaturated aqueous solution of NH₄Cl and AcOEt. After separation, theorganic layer was dried, partially evaporated and filtered. The filtratewas purified by flash chromatography on silica gel(AcOEt/hexane:50/50□75/25) to form a material which after a secondcolumn purification (MeOH/CH₂Cl₂:5/95) afford the title compound XXVIa(400 mg, 1.59 mmol, 31% yield).

Step 2: N-(2-Amino-phenyl)-4-(3-oxo-3-phenyl-propyl)-benzamide (XXVIIa)

The title compound XXVIIa was obtained from XXVIa in one step followingthe same procedure as Example 1, step 2 (Scheme 1). ¹H NMR (300 MHz,DMSO-d₆) δ(ppm): 9.59 (s, 1H); 8.00 (d, J=7.5 Hz, 2H); 7.90 (d, J=7.9Hz, 2H), 7.64 (t, J=7.5 Hz, 2H), 7.43 (d, J=7.9 Hz, 2H), 7.16 (d, J=7.5Hz, 1H), 6.97 (t, J=7.0 Hz, 1H), 6.78 (d, J=7.0 Hz, 1H), 6.59 (t, J=7.5Hz, 1H), 4.88 (bs, 2H), 3.44 (t, J=7.3 Hz, 2H), 3.03 (t, J=7.3 Hz, 2H).

Example 44 N-(2-Amino-phenyl)-4-(3-phenyl-propyl)-benzamide (XXIXa)

Step 1: 4-(3-Phenyl-propyl)-benzoic acid (XXVIIIa)

A stirred solution at room temperature of XXIIIa (1.34 g, 5.31 mmol) in25 ml DMA was hydrogenated over 10% Pd/C (600 mg, Degussa type) at 1 atmfor 3 h. After removal of the catalyst by filtration through celite pad,the solution was concentrated and the residue was treated with water.After precipitation, the suspension was filtered off, rinsed with H₂O,and dried to afford the title compound XXVIIIa (1.13 g, 4.72 mmol, 89%yield).

Step 2: N-(2-Amino-phenyl)-4-(3-phenyl-propyl)-benzamide (XXIXa)

The title compound XXIXa was obtained from XXVIIIa in one step followingthe same procedure as Example 1, step 2 (Scheme 1). ¹H NMR (300 MHz,DMSO-d₆) δ(ppm): 9.60 (s, 1H); 7.91 (d, J=7.9 Hz, 2H); 7.34 (d, J=8.4Hz, 2H), 7.28 (d, J=7.5 Hz, 2H), 7.23-7.15 (m, 4H), 6.97 (t, J=7.0 Hz,1H), 6.78 (d, J=7.5 Hz, 1H), 6.59 (t, J=7.3 Hz, 1H), 4.88 (bs, 2H),2.71-2.59 (m, 4H), 1.92 (m, 2H).

Example 45N-(2-Amino-phenyl)-4-[3-(1,3-dihydro-isoindol-2-yl)-propenyl]-benzamide(XXXa)

Step 1: Methyl 4-trimethylsilanylethynyl-benzoate (XXXI)

A stirred solution at room temperature of methyl 4-bromobenzoate (8.84g, 41.11 mmol), Pd(PPh₃)₂Cl₂ (840 mg, 1.20 mmol) and Cul (455 mg, 2.39mmol) in anhydrous THF (200 ml) was saturated with nitrogen for 15 min.Then, the solution under nitrogen was cooled down at 0° C., andtrimethylsilylacetylene (7.2 ml, 50.91 mmol) and triethylamine (22 ml,157.8 mmol) were added successively. The reaction mixture was allowed towarm up at room temperature. After 2 h, Pd(PPh₃)₂Cl₂ (100 mg) and Cul(80 mg) and trimethylsilylacetylene (0.5 ml) were added again, and thereaction mixture was stirred overnight. Then, the reaction mixture wasdiluted with AcOEt and successively washed with a saturated aqueoussolution of NH₄Cl and brine, dried over MgSO₄, filtered andconcentrated. The crude residue was then purified by flashchromatography on silica gel (AcOEt/hexane:5/95□10/90) to afford thetitle compound XXXI (9.05 g, 38.95 mmol, 94% yield) as a yellow stickysolid. ¹H NMR: (400 MHz, CDCl₃) δ(ppm): AB system (δ_(A)=7.67,δ_(B)=7.22, J_(AB)=8.5 Hz, 4H), 3.63 (s, 3H), 0.00 (s, 9H).

Step 2: Methyl 4-ethynyl-benzoate (XXXII)

To a stirred solution at 0° C. under nitrogen of XXXI (9.05 g, 38.95mmol) in MeOH (280 ml) was added potassium carbonate (1.62 g, 11.72mmol). After 3 h, the reaction mixture was concentrated and directlypurified by flash chromatography on silica gel (CH₂Cl₂:100) to affordthe title compound XXXII (6.16 g, 38.46 mmol, 98% yield) as a paleyellow solid. ¹H NMR: (400 MHz, CDCl₃) δ (ppm): AB system (δ_(A)=7.98,δ_(B)=7.54, J_(AB)=8.6 Hz, 4H), 3.93 (s, 3H), 3.24 (s, 1H).

Step 3: β-(4-methoxycarbonyl)-styrylboronic acid (XXXIII)

To a stirred solution at room temperature under nitrogen of XXXII (6.16g, 38.46 mmol) in anhydrous THF (15 ml) was added catecholborane (4.52ml, 42.80 mmol). The reaction mixture was heated to 70° C. for 4 h, andcathecholborane (2 ml) was added again. After 1.5 h, the reactionmixture was allowed to cool down at room temperature, and an aqueoussolution of 2N HCl (50 ml) was added and stirred overnight. Then, it wasconcentrated on the Rotavap, filtered off and the cake was triturated intoluene. After filtration, the intermediate solid was dissolved in THF(50 ml) and an aqueous solution of 2N HCl (150 ml) was added. Theresulting suspension was warmed to 40° C. for overnight, filtered off,rinsed with water, air-dried and dried under vacuum to afford the titlecompound XXXIII (3.10 g, 15.05 mmol, 39% yield) as an off-white fluffysolid. ¹H NMR: (400 MHz, DMSO-d₆) δ (ppm): AB system (δ_(A)=7.96,δ_(B)=7.63, J_(AB)=8.4 Hz, 4H), 7.94 (s, 2H), 7.32 (d, J=18.2 Hz, 1H),6.30 (d, J=18.2 Hz, 1H), 3.88 (s, 3H).

Step 4: Methyl 4-[3-(1,3-Dihydro-isoindol-2-yl)-propenyl]-benzoate(XXXIVa)

To a stirred solution pre-heated at 90° C. for 15 min under nitrogen ofisoindoline (116 mg, 0.97 mmol) and paraformaldehyde (32 mg, 1.07 mmol)in anhydrous 1,4-dioxane (10 ml) was added XXXIII (245 mg, 1.17 mmol).After stirring at 90° C. for overnight, the reaction mixture was allowedto cool down to room temperature, an aqueous solution of 2N HCl (30 ml)was added and shacked for 30 min. Then, the aqueous mixture wasextracted with Et₂O, basified with 2N NaOH (50 ml), and extracted withCH₂Cl₂. The combined dichoromethane layer was dried over MgSO₄, filteredand concentrated. The crude residue was then purified by flashchromatography on silica gel (MeOH/CH₂Cl₂:5/95) to afford the titlecompound XXXIVa (135 mg, 0.46 mmol, 48% yield) as an off-white solid. ¹HNMR: (400 MHz, DMSO-d₆) δ (ppm): AB system (δ_(A)=7.93, δ_(B)=7.64,J_(AB)=8.4 Hz, 4H), 7.29-7.17 (m,4H), 6.75 (d, J=15.8 Hz, 1H), 6.62 (dt,J=16.0, 6.3 Hz, 1H), 3.94 (s, 4H), 3.88 (s, 3H), 3.55 (dd, J=6.1, 1.0Hz, 2H).

Step 5:N-(2-Amino-phenyl)-4-[3-(1,3-dihydro-isoindol-2-yl)-propenyl]-benzamide(XXXa)

The title compound XXXa was obtained from XXXIVa in two steps followingthe same procedure as Example 11, steps 5 and 6 (Scheme 3). ¹H NMR (400MHz, DMSO-d₆) δ(ppm): 9.67 (s, 1H), AB system (δ_(A)=7.98, δ_(B)=7.63,J_(AB)=8.3 Hz, 4H), 7.30-7.15 (m,5H), 7.00 (td, J=7.6, 1.5 Hz, 1H), 6.81(dd, J=8.0, 1.4 Hz, 1H), 6.75 (d, J=15.8 Hz, 1H), 6.66-6.56 (m, 2H),4.93 (s, 2H), 3.95 (s, 4H), 3.56 (dd, J=6.2, 0.9 Hz, 2H).

Examples 46-49

Examples 46 to 49 (compounds XXXb-XXXe) were prepared using the sameprocedure as described for compound XXXa of Example 45 (Scheme 11).TABLE 6 XXX

Cmpd RX- NAME Characterization Scheme XXXb

N-(2-Amino-phenyl)-4-[3- (4-benzyl-piperazin-1- yl)-propenyl]-benzamide¹H NMR (400 MHz, CDCl₃) δ (ppm): 7.84 (d, J = 8.0 Hz, 3H), 7.46 (d, J =8.0 Hz, 2H), 7.35-7.22 (m, 6H), 7.09 (td, J = 7.6, 1.6 Hz, 1H),6.88-6.81 (m, 2H), 6.58 (d, J = 15.6 Hz, 1H), 6.41 (dt, J = 15.6, 6.8Hz, 1H), 3.88 (bs, 2H), 3.57 (s, 2H), 3.24 (d, J = 6.8 Hz, 2H), 2.59(bs, 8H). 11 XXXc

N-(2-Amino-phenyl)-4-[3- (4-pyridin-2-yl-piperazin-1-yl)-propenyl]-benzamide ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 9.63 (s,1H), 8.09-8.08 (m, 1H), 7.93 (d, J = 8.0 Hz, 2H), 8.0 (d, J = 2.5 Hz,1H), 7.51 (t, J = 7.3 Hz, 1H), 7.14 (d, J = 7.6, 1H), 6.95 (td, J = 7.6,1.2 Hz, 1H), 6.81 (d, J = 8.6, 1H), 6.76 (dd, J = 8.0, 1.4 Hz, 1H),6.69-6.60 (m, 2H), # 6.58 (td, J = 7.6, 1.3 Hz, 1H), 6.49 (dt, J = 16.0,6.5 Hz, 1H), 4.89 (s, 2H), 3.48-3.15 (m, 6H), 2.53-2.45 (m, 4H). 11 XXXd

N-(2-Amino-phenyl)-4-[3- (benzyl-methyl-amino)- propenyl]-benzamide ¹HNMR (400 MHz, DMSO-d₆) δ (ppm) 9.66 (s, 1H), 7.96 (bd, J = 8.2 Hz, 2H),7.60 (bd, J = 8.2 Hz, 2H), 7.34 (s, 2H), 7.36 (d, J = 1.2 Hz, 2H),7.30-7.25 (m, 1H), 7.18 (d, J = 7.0 Hz 1H), 6.99 (td, J = 7.6, 1.5 Hz,1H), 6.76 (dd, J = 7.9, 1.5 Hz, 1H), 6.68 (d, J = 15.8, 1H), 6.62 (td, J= 7.5, # 1.4 Hz, 1H), 6.54 (dt, J = 16.2, 6.5 Hz, 1H), 4.92 (bs, 2H),3.57 (s, 2H), 3.22 (d, J = 6.3 Hz, 2H), 2.20 (s, 3H). 11 XXXe

N-(2-Amino-phenyl)-4-[3- (indan-2-ylamino)- propenyl]-benzamide ¹H NMR(400 MHz, DMSO-d₆) δ (ppm) 9.71 (s, 1H), 8.02 (d, J = 8.2 Hz, 2H), 7.64(d, J = 8.4 Hz, 2H), 7.32-7.22 (m, 4H), 7.18 (d, J = 7.6 Hz, 1H), 7.00(t, J = 8.1 Hz, 1H), 6.87 (d, J = 16.0 Hz, 1H), 6.80 (d, J = 9.2 Hz,1H), 6.63 (t, J = 7.7 Hz, 1H), 6.50 (dt, J = 15.8, 6.7 Hz, 1H), 4.93 #(bs, 2H), 4.13 (m, 1H), 3.87 (d, J =7.6 Hz, 2H), 3.37-3.32 (m, 2H), 3.14(d, J = 7.6 Hz, 2H). 11

Example 50 Inhibition of Histone Deacetylase Enzymatc Activity

1. Human HDAC-1: Assay 1

HDAC inhibitors were screened against a cloned recombinant human HDAC-1enzyme expressed and purified from a Baculovirus insect cell expressionsystem. For deacetylase assays, 20,000 cpm of the [³H]-metabolicallylabeled acetylated histone substrate (M. Yoshida et al., J. Biol. Chem.265(28): 17174-17179 (1990)) was incubated with 30 μg of the clonedrecombinant hHDAC-1 for 10 minutes at 37° C. The reaction was stopped byadding acetic acid (0.04 M, final concentration) and HCl (250 mM, finalconcentration). The mixture was extracted with ethyl acetate and thereleased [³H]-acetic acid was quantified by scintillation counting. Forinhibition studies, the enzyme was preincubated with compounds at 4° C.for 30 minutes prior to initiation of the enzymatic assay. IC₅₀ valuesfor HDAC enzyme inhibitors were determined by performing dose responsecurves with individual compounds and determining the concentration ofinhibitor producing fifty percent of the maximal inhibition. IC₅₀ valuesfor representative compounds assayed using this procedure are presentedin the third column of Tables 7-10 (excepting bracketed data).

2. Human HDAC-1: Assay 2

In the alternative, the following protocol was used to assay thecompounds of the invention. In the assay, the buffer used was 25 mMHEPES, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂ and the subtrate wasBoc-Lys(Ac)-AMC in a 50 mM stock solution in DMSO. The enzyme stocksolution was 4.08 μg/mL in buffer.

The compounds were pre-incubated (2 μl in DMSO diluted to 13 μl inbuffer for transfer to assay plate) with enzyme (20 μl of 4.08 μg/ml)for 10 minutes at room temperature (35 μl pre-incubation volume). Themixture was pre-incubated for 5 minutes at room temperature. Thereaction was started by bringing the temperature to 37° C. and adding 16μl substrate. Total reaction volume was 50 μl. The reaction was stoppedafter 20 minutes by addition of 50 μl developer, prepared as directed byBiomol (Fluor-de-Lys developer, Cat. # KI-105). A plate was incubated inthe dark for 10 minutes at room temperature before reading (λ_(EX)=360nm, λ_(Ex)=470 nm, Cutoff filter at 435 nm).

IC₅₀ values for representative compounds assayed using this procedureare presented in the third column of Table 9 (bracketed [ ] data).

3. MTT Assay

HCT116 cells (2000/well) were plated into 96-well tissue culture platesone day before compound treatment. Compounds at various concentrationswere added to the cells. The cells were incubated for 72 hours at 37° C.in 5% CO₂ incubator. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, Sigma) was added at a final concentration of 0.5mg/ml and incubated with the cells for 4 hours before one volume ofsolubilization buffer (50% N,N-dimethylformamide, 20% SDS, pH 4.7) wasadded onto the cultured cells. After overnight incubation, solubilizeddye was quantified by colorimetric reading at 570 nM using a referenceat 630 nM using an MR700 plate reader (Dynatech Laboratories Inc.). ODvalues were converted to cell numbers according to a standard growthcurve of the relevant cell line. The concentration which reduces cellnumbers to 50% of that of solvent treated cells is determined as MTTIC₅₀. IC₅₀ values for representative compounds are presented in thefourth column of Tables 7-10.

4. Histone H4 Acetylation in Whole Cells by Immunoblots

T24 human bladder cancer cells growing in culture were incubated withHDAC inhibitors for 16 h. Histones were extracted from the cells afterthe culture period as described by M. Yoshida et al. (J. Biol. Chem.265(28): 17174-17179 (1990)). 20 g of total histone protein was loadedonto SDS/PAGE and transferred to nitrocellulose membranes. Membraneswere probed with polyclonal antibodies specific for acetylated histoneH-4 (Upstate Biotech Inc.), followed by horse radish peroxidaseconjugated secondary antibodies (Sigma). Enhanced Chemiluminescence(ECL) (Amersham) detection was performed using Kodak films (EastmanKodak). Acetylated H-4 signal was quantified by densitometry.Representative data are presented in the fifth column of Table 7-10.Data are presented as the concentration effective for reducing theacetylated H-4 signal by 50% (EC₅₀). TABLE 7

Human HDAC-1 MTT(HCT116) H4 Ac (T24) Cmpd Structure IC₅₀ (μM) IC₅₀ (μM)EC₅₀ (μM) Ia

5 1 na Ib

2 0.9 5 Ic

3 0.4 9999 Id

3 0.6 3 Ie

6 9 na If

4 5 na Ig

3 0.7 9999 Ih

5 1 5 Ii

3 1 3 Ij

4 0.7 5(na = non available, 9999 = >25 mM)

TABLE 8

Human HDAC-1 MTT(HCT116) H4 Ac (T24) Cmpd Structure IC₅₀ (μM) IC₅₀ (μM)EC₅₀ (μM) Va

5 3 na Vb

3 0.07 1 Vc

3 0.9 2 Vd

1 0.4 1 Ve

20 6 na Vf

5 0.3 1 Vg

10 5 na Vh

3 1 10 Vi

4 1 3 Vj

1 0.3 1 Vk

13 2 na Vl

12 4 na Vm

11 6 na Vn

7 3 na Vo

15 4 na V9

13 2 na(na = non available, 9999 = >25 mM)

TABLE 9

Human HDAC-1 MTT(HCT116) H4 Ac (T24) Cmpd Structure IC₅₀ (μM) IC₅₀ (μM)EC₅₀ (μM) XVa

2 1 1 XVb

4 0.1 3 XVc

0.8 1 <1 XVd

4 0.3 3 XVe

2 0.5 1 XVf

3 1 2 XVg

12 5 na XXIIh

3 3 na XVh

4 0.7 2 XVi

1 1 4 XVj

2 0.2 <1 XXXa

[0.26] 1 4 XXXb

4 1 2 XXXc

[0.82] 0.5 2 XXXd

[0.17] 2 na XXXe

[0.79] 2 na(na = non available, 9999 = >25 mM)

TABLE 10

Human HDAC-1 MTT(HCT116) H4 Ac (T24) Cmpd Structure IC₅₀ (μM) IC₅₀ (μM)EC₅₀ (μM) XXIVa

4 0.4 3 XXIVb

8 0.1 3 XXIVc

13 0.6 5 XXIVd

4 0.3 <5 XXIVe

3 0.8 5 XXIVf

10 0.3 3 XXVIIa

2 0.6 2 XXIXa

2 0.6 na(na = non available, 9999 = >25 mM)

Example 51 Antineoplastic Effects of Histone Deacetylase Inhibitors onHuman Tumor Xenografts In Vivo

Eight to ten week old female CD1 nude mice (Taconic Labs, GreatBarrington, N.Y.) were injected subcutaneously in the flank area with2×10⁶ preconditioned HCT116 human colorectal carcinoma cells.Preconditioning of these cells was done by a minimum of threeconsecutive tumor transplantations in the same strain of nude mice.Subsequently, tumor fragments of approximately 30 mgs were excised andimplanted subcutaneously in mice, in the left flank area, under Foreneanesthesia (Abbott Labs, Geneva, Switzerland). When the tumors reached amean volume of 100 mm³, the mice were treated intravenously,subcutaneously, or intraperitoneally by daily injection, with a solutionof the histone deacetylase inhibitor in an appropriate vehicle, such asPBS, DMSO/water, or Tween 80/water, at a starting dose of 10 mg/kg. Theoptimal dose of the HDAC inhibitor was established by dose responseexperiments according to standard protocols. Tumor volume was calculatedevery second day post infusion according to standard methods (e.g.,Meyer et al., Int. J. Cancer 43: 851-856 (1989)). Treatment with theHDAC inhibitors according to the invention caused a significantreduction in tumor weight and volume relative to controls treated withvehicle only (i.e., no HDAC inhibitor); a subset of these compoundsshowed toxicity. The results for compound XVj as an example aredisplayed in FIG. 1.

1. A compound of the following formula:

or pharmaceutically acceptable salt thereof, wherein Ar is aryl orheteroaryl, each of which is optionally substituted with from 1 to 3substituents.
 2. The compound of claim 1 wherein Ar is aryl orpyridinyl.
 3. The compound of claim 1 wherein Ar is phenyl.
 4. Thecompound of claim 1 wherein Ar is substituted with 1-3 substituentsselected from the group consisting of halo, C₁-C₆-hydrocarbyl optionallysubstituted with halo, C₁-C₆-hydrocarbyloxy optionally substituted withhalo.
 5. The compound of claim 1 wherein Ar is selected from one of thefollowing:


6. A compound of the following formula:

or pharmaceutically acceptable salt thereof, wherein X is —N(R¹)—, —O—,or —S—; or X is a nitrogen-containing heterocyclyl in which a nitrogenis covalently bound to the adjacent carbonyl in structure V and isoptionally substituted with from 1 to 3 substituents; and R andR¹independently are —H, or optionally substituted a) C₁-C₆-hydrocarbylor b) R²-L-, wherein R² is aryl or heteroaryl, L isC₀-C₆-hydrocarbyl-L¹-C₀-C₆-hydrocarbyl, and L¹ is a covalent bond, —O—,—S—, or —NH—.
 7. The compound according to claim 6 wherein X is —NH—,—O—, morphilin-4-yl, piperidin-1-yl, piperizin-1-yl, or pyrrolidin-1-yl.8. The compound according to claim 6 wherein X is —N(R¹)— wherein R¹ isoptionally substituted methyl or ethyl.
 9. The compound according toclaim 6 wherein X is —N(R¹)— wherein R¹ is cyanoethyl orpyridinylmethyl.
 10. The compound according to claim 6 wherein X is—N(R¹)— wherein R is R²-L- wherein R² is phenyl, pyridinyl, indyl, orindolyl and L is a covalent bond, methyl, ethyl, or oxyethyl.
 11. Thecompound according to claim 6 wherein the combination of R—X— isselected from the following:


12. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein Y is —N(R⁴)—,—O—, —S—, —N(R⁴)SO₂—, —SO₂—N(R⁴) —, —SO₂—, —N(R⁴)—C(O)—, —C(O)—N(R⁴)—,—NHC(O)NH—, —N(R⁴)C(O)O—, —OC(O)N(R⁴)—, or a covalent bond, and R¹, R²,and R³ independently are —H or R¹—C₀-C₆-hydrocarbyl wherein R^(a) is —Hor R^(a) is aryl or heteroaryl, each of which is optionally substitutedwith from 1 to 3 substituents. R⁴ is —H, —C(O)—R^(b), —C(O)O—R^(b),—C(O)NH—R^(b), or R—C₀-C₆-hydrocarbyl wherein R^(b) is —H or—C₁-C₆-hydrocarbyl, and R^(c) is —H, or aryl or heteroaryl each of whichis optionally substituted with from 1 to 3 substituents.
 13. Thecompound according to claim 12 wherein R² and R³ are both —H.
 14. Thecompound according to claim 12 wherein Y is —NH—, —SO₂—NH—, or —N(R⁴)—wherein R⁴ is —C(O)O—C₁-C₆-hydrocarbyl.
 15. The compound according toclaim 12 wherein R¹ is aryl, benzothiazolyl, pyrimidinyl, triazolyl,benzodioxolenyl, or pyridinyl, each of which is optionally substitutedwith from 1 to 3 substituents.
 16. The compound according to claim 15wherein R¹ is substituted with from 1-3 substituents independentlyselected from C1-C₆-hydrocarbyl, C₁-C₆-hydrocarbyloxy, halo, methylthio,and acetyl.
 17. The compound according to claim 12 selected from thefollowing:


18. A compound of formula:

or a pharmaceutically acceptable salt thereof, wherein Ar¹ is aryl orheteroaryl optionally substituted with from 1-3 substituentsindependently selected from —NO₂, CH₃O—, and morpholinyl (e.g.,morpholin-4-yl).
 19. The compound according to claim 18 wherein Ar¹ isaryl optionally substituted with from 1-3 substituents independentlyselected from —NO₂, CH₃O—, and morpholinyl (e.g., morpholin-4-yl). 20.The compound according to claim 18 wherein Ar¹ is phenyl optionallysubstituted with from 1-3 substituents independently selected from —NO₂,CH₃O—, and morpholinyl (e.g., morpholin-4-yl).
 21. The compoundaccording to claim 18 selected from:


22. A composition comprising a compound according to claim 1 and apharmaceutically acceptable carrier, excipient, or diluent.
 23. A methodof inhibiting histone deacetylase in a cell, comprising contacting acell in which inhibition of histone deacetylase is desired with aninhibitor of histone deacetylase according to claim
 1. 24. A method oftreating a mammal suffering from a cell proliferative disease orcondition a therapeutically effective amount of a composition accordingto claim
 22. 25. The method according to claim 24 wherein the mammal isa human.
 26. A composition comprising a compound according to claim 6and a pharmaceutically acceptable carrier, excipient, or diluent.
 27. Amethod of inhibiting histone deacetylase in a cell, comprisingcontacting a cell in which inhibition of histone deacetylase is desiredwith an inhibitor of histone deacetylase according to claim
 6. 28. Amethod of treating a mammal suffering from a cell proliferative diseaseor condition a therapeutically effective amount of a compositionaccording to claim
 26. 29. The method according to claim 28 wherein themammal is a human.
 30. A composition comprising a compound according toclaim 12 and a pharmaceutically acceptable carrier, excipient, ordiluent.
 31. A method of inhibiting histone deacetylase in a cell,comprising contacting a cell in which inhibition of histone deacetylaseis desired with an inhibitor of histone deacetylase according to claim12.
 32. A method of treating a mammal suffering from a cellproliferative disease or condition a therapeutically effective amount ofa composition according to claim
 30. 33. The method according to claim32 wherein the mammal is a human.
 34. A composition comprising acompound according to claim 18 and a pharmaceutically acceptablecarrier, excipient, or diluent.
 35. A method of inhibiting histonedeacetylase in a cell, comprising contacting a cell in which inhibitionof histone deacetylase is desired with an inhibitor of histonedeacetylase according to claim
 18. 36. A method of treating a mammalsuffering from a cell proliferative disease or condition atherapeutically effective amount of a composition according to claim 34.37. The method according to claim 36 wherein the mammal is a human.